Kits and reaction mixtures containing modified probe molecules

ABSTRACT

The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.

This application is a continuation of application Ser. No. 09/565,427,filed May 5, 2000, now U.S. Pat. No. 7,070,925, the contents of whichare hereby incorporated by reference herein, which is a continuation ofapplication Ser. No. 08/893,300, filed Jul. 15, 1997, now U.S. Pat. No.6,130,038, which claims the benefit of U.S. Provisional Application No.60/021,818, filed Jul. 16 1996.

FIELD OF THE INVENTION

This invention pertains to methods and compositions for detecting andamplifying nucleic acid sequences using oligonucleotides which containone or more nucleotides having a modification or modifications resultingin increased target affinity. Such oligonucleotides have beenunexpectedly discovered to hybridize to a target nucleic acid at asignificantly greater rate than a corresponding unmodifiedoligonucleotide hybridizes to the same target. As a result, the methodsand compositions of the present invention offer advantages forapplications employing nucleic acid hybridization, such as medical andveterinary diagnostics, food testing and forensics.

BACKGROUND OF THE INVENTION

In recent years, nucleic acid hybridization has become an increasinglyimportant means of identifying, measuring and detecting the presence ofparticular nucleic acids in a given sample. Thus, for example, thefields of medical diagnostics, environmental and food testing, andforensics have all benefitted from the use of nucleic acid hybridizationas a rapid, simple and extraordinarily accurate way of testing for thepresence or absence of given biological contaminants or microorganismsin a sample.

Most nucleic acid hybridization schemes have features in common. Onesuch typical feature is the use of single-stranded nucleic acid probes(or denatured double-stranded probes) having a defined or knownnucleotide sequence. Probe molecules may be derived from biologicalsources, such as genomic DNA or RNA, or may be enzymaticallysynthesized, either in a prokaryotic or eukaryotic host cell or invitro. Presently, most nucleic acid probes in common use areoligonucleotide probes made using chemical synthetic methods (“syntheticoligonucleotides”). One such synthetic method is automated sequentialaddition of 3′-activated, protected nucleotides to the 5′ end of agrowing, solid phase-bound oligonucleotide chain, followed by cleavageof the completed oligonucleotide from the support and deprotection. See,e.g., Eckstein, Oligonucleotides & Analogues: A Practical Approach(1991).

Synthetic oligonucleotides for use as hybridization probes are typicallydeoxyribonucleotides having a nucleotide sequence complementary to anucleotide sequence of the nucleic acid to be detected. DNAoligonucleotides are classically preferred for a number of reasons.Among these is the greater stability DNA has to enzymatic hydrolysisupon exposure to common samples, due to the almost ubiquitous presencein samples of various RNAses. RNA is also known to be less chemicallystable than DNA, e.g., RNA degradation is facilitated by the presence ofbase, heavy metals. And compared to RNA, DNA is less prone to assumestable secondary structures under assay conditions. Such secondarystructures can render oligonucleotides unavailable for inter-molecularhybridization. Nevertheless, RNA oligonucleotides may be used, eventhough they are less preferred.

Nucleic acid hybridization exploits the ability of single-strandednucleic acids to form stable hybrids with corresponding regions ofnucleic acid strands having complementary nucleotide sequences. Suchhybrids usually consist of double-stranded duplexes, althoughtriple-stranded structures are also known. Generally speaking,single-strands of DNA or RNA are formed from nucleotides containing thebases adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U),or inosine (I). The single-stranded chains may hybridize to form adouble-stranded structure held together by hydrogen bonds between pairsof complementary bases. Generally, A is hydrogen bonded to T or U, whileG or I is hydrogen bonded to C. Along the double-stranded chain,classical base pairs of AT or AU, TA or UA, GC, or CG are present.Additionally, some mismatched base pairs (e.g., AG, GU) may be present.Under appropriate hybridization conditions, DNA/DNA, RNA/DNA, or RNA/RNAhybrids can form.

By “complementary” is meant that the nucleotide sequences ofcorresponding regions of two single-stranded nucleic acids, or twodifferent regions of the same single-stranded nucleic acid, have anucleotide base composition that allows the single strands to hybridizetogether in a stable double-stranded hydrogen-bonded region understringent hybridization conditions. When a contiguous sequence ofnucleotides of one single stranded region is able to form a series of“canonical” hydrogen-bonded base pairs with an analogous sequence ofnucleotides of the other single-stranded region, such that A is pairedwith U or T, and C is paired with G, the nucleotides sequences are“perfectly” complementary.

The extreme specificity of nucleic acid hybridization, which under somecircumstances can allow the discrimination of nucleic acids differing byas little as one base, has allowed the development ofhybridization-based assays of samples containing specificmicroorganisms, nucleic acids bearing given genetic markers, tissue,biological fluids and the like. Such assays are often able to identifynucleic acids belonging to particular species of microorganisms in asample containing other, closely-related species. Nucleic acidhybridization assays can also specifically detect or identify certainindividuals, or groups of individuals, within a species, such as in theforensic use of RFLP (restriction fragment length polymorphism) and PCR(polymerase chain reaction) testing of samples of human origin.

The use of oligonucleotides as a diagnostic tool in nucleic acidhybridization testing often involves, but need not involve, the use of areporting group or “label” which is joined to the oligonucleotide probemolecule, or both the probe and the target. Such a reporter group moietymay include, for example, a radioisotope, chemiluminescent orfluorescent agent, or enzyme joined to the oligonucleotide. The label isemployed to render the probe capable of detection, particularly when theprobe is hybridized to the target nucleic acid.

The majority of assay methods employing nucleic acids utilize a physicalseparation step in order to separate the probe:analyte hybrid fromunbound probe. These assay methods are called “heterogeneous” assays. Innucleic acid hybridization assays, an analyte molecule is the targetnucleic acid species sought to be detected, quantitated and/oridentified. A “hybrid” is a partly or wholly double-stranded nucleicacid comprising two single-stranded nucleic acids, such as a probe and atarget nucleic acid, having a region of complementarity resulting inintermolecular hydrogen bonding under assay and/or amplificationconditions.

Assay methods utilizing a physical separation step include methodsemploying a solid-phase matrix, such as glass, minerals or polymericmaterials, in the separation process. The separation may involvepreferentially binding the probe:analyte complex to the solid phasematrix, while allowing the unassociated probe molecules to remain in aliquid phase. Such binding may be non-specific, as, for example, in thecase of hydroxyapatite, or specific, for example, throughsequence-specific interaction of the target nucleic acid with a“capture” probe which is directly or indirectly immobilized on the solidsupport. In any such case, the amount of probe remaining bound to thesolid phase support after a washing step is proportional to the amountof analyte in the sample.

Alternatively, the assay may involve preferentially binding theunhybridized probe while leaving the hybrid to remain in the liquidphase. In this case the amount of probe in the liquid phase after awashing step is proportional to the amount of analyte in the originalsample. When the probe is a nucleic acid or oligonucleotide, the solidsupport can include, without limitation, an adsorbent such ashydroxyapatite, a polycationic moiety, a hydrophobic or “reverse phase”material, an ion-exchange matrix, such as DEAE, a gel filtration matrix,or a combination of one or more of these solid phase materials. Thesolid support may contain one or more oligonucleotides, or otherspecific binding moiety, to capture, directly or indirectly, probe,target, or both. In the case of media, such as gel filtration,polyacrylamide gel or agarose gel, the separation is not due to bindingof the oligonucleotide but is caused by molecular sieving of differentlysized or shaped molecules. In the latter two cases, separation may bedriven electrophoretically by application of an electrical currentthrough the gel causing the differential migration through the gel ofnucleic acids of different sizes or shapes, such as double-stranded andsingle-stranded nucleic acids.

A heterogeneous assay method may also involve binding the probe to asolid-phase matrix prior to addition of a sample suspected of containingthe analyte of interest. The sample can be contacted with the labelunder conditions which would cause the desired nucleic acid to belabeled, if present in the sample mixture. The solid phase matrix may bederivatized or activated so that a covalent bond is formed between theprobe and the matrix. Alternatively, the probe may be bound to thematrix through strong non-covalent interactions, including, withoutlimitation, the following interactions: ionic, hydrophobic,reverse-phase, immunobinding, chelating, and enzyme-substrate. After thematrix-bound probe is exposed to the labeled nucleic acid underconditions allowing the formation of a hybrid, the separation step isaccomplished by washing the solid-phase matrix free of any unbound,labeled analyte. Conversely, the analyte can be bound to the solid phasematrix and contacted with labeled probe, with the excess free probewashed from the matrix before detection of the label.

Yet another type of assay system is termed “homogeneous assay.”Homogenous assays can generally take place in solution, without a solidphase separation step, and commonly exploit chemical differences betweenthe free probe and the analyte:probe complex. An example of an assaysystem which can be used in a homogenous or heterogeneous format is thehybridization protection assay (HPA) disclosed in Arnold, et al., U.S.Pat. No. 5,283,174, in which a probe is linked to a chemiluminescentmoiety, contacted with an analyte and then subjected to selectivechemical degradation or a detectable change in stability underconditions which alter the chemiluminescent reagent bound or joined tounhybridized probe, without altering the chemiluminescent reagent boundor joined to an analyte:probe conjugate. Subsequent initiation of achemiluminescent reaction causes the hybrid-associated label to emitlight. This patent enjoys common ownership with the present applicationand is expressly incorporated by reference herein.

Competition assays, in which a labeled probe or analyte competes forbinding with its unlabeled analog, are also commonly used in aheterogeneous format. Depending on how the system is designed, eitherthe amount of bound, labeled probe or the amount of unbound, labeledprobe can be correlated with the amount of analyte in a sample. However,such an assay can also be used in a homogeneous format without aphysical separation step, or in a format incorporating elements of botha homogeneous and a heterogeneous assay.

The assay methods described herein are merely illustrative and shouldnot be understood as exhausting the assay formats employing nucleicacids known to those of skill in the art.

Nucleic acid hybridization has been utilized in methods aimed at usingoligonucleotides as therapeutic agents to modify or inhibit geneexpression within living organisms. In an example of such utilization,oligonucleotide “antisense” agents can be targeted specifically to anmRNA species encoding a deleterious gene product, such as a viralprotein or an oncogene. See, e.g., Zamecnik and Stephenson, 75 Proc.Nat'l Acad. Sci. (USA), 280-284 (1978); Stephenson and Zamecnik, 75Proc. Nat'l Acad. Sci. (USA), 285-288 (1978); and Tullis, U.S. Pat. No.5,023,243. Although Applicant does not wish to be bound by theory, it isthought that the RNA:DNA duplex which results from the binding of theantisense oligonucleotide to RNA targets may serve as a substrate forRNAse H, an RNA-degrading enzyme present in most cells and specific forRNA contained in an RNA:DNA duplex. According to this model, the targetRNA molecule is destroyed through hybridization to the antisenseoligonucleotide. Variations of this general strategy exist, wherein, forexample, the oligonucleotide has a structure conferring an enzymaticactivity on the oligonucleotide, such as the RNAse activity of so-calledribozymes. See, e.g., Goodchild, PCT Publication No. WO93/15194.

Because therapeutic antisense oligonucleotides are primarily designed tofunction in vivo, formulations for the delivery of such agents must notsignificantly inhibit normal cellular function. Thus, nucleaseinhibitors, which can sometimes be included in in vitro diagnostic teststo prevent oligonucleotide degradation, are not suitable for use invivo. This fact has resulted in the design of various oligonucleotidesmodified at the internucleotide linkage, at the base or sugar moieties,or at combinations of these sites to have greater nuclease resistancethan unmodified DNA.

Thus, a number of oligonucleotide derivatives have been made havingmodifications at the nitrogenous base, including replacement of theamino group at the 6 position of adenosine by hydrogen to yield purine;substitution of the 6-keto oxygen of guanosine with hydrogen to yield2-amino purine, or with sulphur to yield 6-thioguanosine, andreplacement of the 4-keto oxygen of thymidine with either sulphur orhydrogen to yield, respectively, 4-thiothymidine or 4-hydrothymidine.All these nucleotide analogues can be used as reactants for thesynthesis of oligonucleotides. See, e.g., Oligonucleotides andAnalogues: A Practical Approach, supra. Other substituted bases areknown in the art. See, e.g., Cook, et al., PCT Publication No.WO92/02258, entitled Nuclease Resistant, Pyrimidine ModifiedOligonucleotides that Detect and Modulate Gene Expression, which isincorporated by reference herein. Base-modified nucleotide derivativescan be commercially obtained for oligonucleotide synthesis.

Similarly, a number of nucleotide derivatives have been reported havingmodifications of the ribofuranosyl or deoxyribofuranosyl moiety. See,e.g., Cook et al., PCT Publication No. WO94/19023 entitled CyclobutylAntisense Oligonucleotides, Methods of Making and Use Thereof; McGee, etal., PCT Publication No. WO94/02501 entitled Novel 2′-O-AlkylNucleosides and Phosphoramidites Processes for the Preparation and UsesThereof; and Cook, PCT Publication No. WO93/13121 entitled Gapped2′-modified Oligonucleotides. These three publications are incorporatedby reference herein.

Most oligonucleotides comprising such modified bases have beenformulated with increased cellular uptake, nuclease resistance, and/orincreased substrate binding in mind. In other words, sucholigonucleotides are described as therapeutic gene-modulating agents.

Nucleic acids having modified nucleotide residues exist in nature. Thus,depending on the type or source, modified bases in RNA can includemethylated or dimethylated bases, deaminated bases, carboxylated bases,thiolated bases and bases having various combinations of thesemodifications. Additionally, 2′-O-alkylated bases are known to bepresent in naturally occurring nucleic acids. See, Adams, TheBiochemistry of the Nucleic Acids, 7,8 (11th ed. 1993).

SUMMARY OF THE INVENTION

This invention concerns diagnostic methods and compositions employingnucleic acid hybridization techniques. Applicant has surprisinglydiscovered that oligonucleotides, comprised of one or more modifiednucleotides, which have increased binding affinity to a target nucleicacid having a complementary nucleotide sequence, will hybridize to thetarget nucleic acid at a faster rate than unmodified oligonucleotides.The inventions described herein are drawn to the use ofoligonucleotides, wholly or partially so modified, in methods involvingtheir use as, for example, hybridization assay probes, amplificationprimers, helper oligonucleotides, and oligonucleotides for the captureand immobilization of desired nucleic acids.

Although the present invention is not to be seen as so limited, inparticularly preferred embodiments the present invention concernsdiagnostic methods utilizing oligonucleotides having nucleotides with 2′modifications to their ribofuranosyl (or deoxyribofuranosyl) moieties.In particular, Applicant has discovered that incorporating nucleotideshaving such modifications as part of synthetic oligonucleotides canprofoundly increase the rate of nucleic acid hybridization, and thepreferential binding of such oligonucleotides, to RNA targets over DNAtargets. A further advantage of these modified oligonucleotides is theirability to efficiently strand invade double-stranded regions ofstructured RNA molecules, especially when the double-stranded RNA duplexis flanked by a single-stranded region of at least about threenucleotide bases. A currently preferred embodiment makes use ofoligonucleotides containing nucleotide analogues having2′-O-methylribofuranosyl moieties linked to a nitrogenous base. Othersubstitutions at the 2′ position of the sugar would be expected, inlight of the present disclosure, to have similar properties so long asthe substitution is not so large as to cause steric inhibition ofhybridization.

Additionally, in light of the discoveries giving rise to the presentinvention, other modifications which increase the T_(m) of a modifiedoligonucleotide:target hybrid would reasonably be expected to contributeto increases in the rate of hybridization as well. Such modificationsmay occur at the 2′ position (or other positions) of thedeoxyribofuranosyl or ribofuranosyl moiety (such as 2′ halidesubstitutions), on the nitrogenous bases (such asN-diisobutylaminomethylidene-5-(1-propynyl)-2′-deoxycytidine; a cytidineanalog, or 5-(1-propynyl)-2′-deoxyuridine); a thymidine analog, or inthe linkage moiety. Thus, while specific reference is made throughoutthis application to 2′ modifications, those of skill in the art willunderstand that other modifications leading to an increased T_(m) of amodified oligonucleotide:target hybrid over a hybrid containing anunmodified oligonucleotide of identical base sequence would be expectedto have similar properties and effects on hybridization kinetics.

While the term T_(m) refers to the temperature at which 50% of apopulation of equal amounts of complementary nucleic acid strands are inthe double-stranded form, throughout this disclosure the “T_(m) of anoligonucleotide” or a nucleic acid (single-stranded) is intended to meanthe T_(m) of an oligonucleotide or nucleic acid in a nucleic acid duplexwith a target, wherein the nucleic acid contains a base sequence regionwhich is exactly complementary to a base sequence region of theoligonucleotide, unless otherwise indicated.

Because the T_(m) of the modified oligonucleotides is higher than thatof corresponding, unmodified oligonucleotides of the same base sequence,the compositions and diagnostic methods described herein enable the useof oligonucleotides and oligonucleotide probes of shorter length thanare otherwise practical for the specific hybridization and detection ofnucleic acid targets (preferably RNA targets). The use of shorteroligonucleotides to specifically bind to target nucleic acids at a giventemperature has additional advantages. For instance, shorteroligonucleotides will generally have a greater ability to discriminateperfectly complementary targets from “mismatched” base sequence regions.Shorter oligonucleotides are also less likely to overlap undesirablebase sequences. Additionally, because of the higher T_(m), the modifiedoligonucleotides can stably hybridize at higher temperatures than theirunmodified counterparts.

The use of higher hybridization temperatures kinetically drives thehybridization reaction, resulting in faster hybridization rates thanwould occur at lower temperatures. Further, the modifiedoligonucleotides used in the methods of the present invention result infaster hybridization rates than the unmodified versions, even when thetemperature is not raised.

An increased hybridization rate leads to several other advantages indiagnostic assays. For example, diagnostic assays conducted inaccordance with the present invention can be conducted more rapidly thanin previously existing hybridization assays. In cases in which theresults of the assay may dictate a course of medical treatment or otheraction, a faster assay result has clear prognostic advantages and mayresult in more effective treatment. Also, owing to faster hybridizationrates and greater affinity of modified oligonucleotides for targets,especially RNA targets, lower concentrations of probe may be used toachieve the same amount of signal. Thus, the assay background (or“noise”) can be reduced and the lower concentration of probe can helpeliminate undesirable cross-reactions with non-target nucleic acids.Additionally, these assays may be run in larger volumes of sample, thusincreasing the sensitivity of the assay.

Thus, hybridization assay probes, amplification oligonucleotides, samplepreparation oligonucleotides and/or helper oligonucleotides can all bedesigned to contain modified bases which have the advantage ofincreasing the rate of target-specific hybridization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, 1B, and 1C provides the IUPAC nomenclature for a sampling ofacridinium esters that may be used as detectable chemiluminescent labelsin the present disclosure.

FIG. 2 shows an arrangement whereby detection of an analyte firstrequires the hybridization of the analyte to a nucleic acid other thanthe probe. According to this arrangement, the probe is unable to bindwith either the analyte or the non-probe nucleic acid before the analytehas hybridized to the non-probe nucleic acid. (Bolded portions representregions of complementary between the analyte and the non-probe nucleicacid.) However, hybridization of the analyte to the non-probe nucleicacid alters the configuration of the non-probe nucleic acid sufficientlyto enable hybridization of the non-probe nucleic acid to the probe, thuspermitting detection of the analyte.

FIG. 3 shows the melting curve of a 2′-O-methyl oligonucleotide probewith either an RNA target or a DNA target (two independent experiments),where melting is shown as an increase in light absorbance at 260 nm(hyperchromatic shift).

FIG. 4 shows hybridization of a single concentration of acridiniumester-labeled deoxy- or 2′-O-methyl oligonucleotides of identical basesequence to varying amounts of a fully complementary RNA target during afixed time of hybridization.

FIG. 5 shows hybridization of varying amounts of acridiniumester-labeled deoxy- or 2′-O-methyl oligonucleotides of identical basesequence to fixed amounts of a fully complementary RNA target during afixed time of hybridization.

FIG. 6 shows hybridization of a fixed amount of acridinium ester-labeleddeoxy- and 2′-O-methyl oligonucleotides of identical base sequence to afixed amount of a fully complementary RNA target for various times ofhybridization.

FIG. 7 shows hybridization of a DNA or 2′-O-methyl oligonucleotide probeto a fully complementary RNA target. The data are plotted according tothe equation ln(1−H)=(k)(C_(o)t), where H is the percent hybridization,k is the hybridization rate constant, C_(o) is the concentration ofprobe, and t is time.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless clearly indicated otherwise, the following terms will have theindicated meanings throughout this specification.

By “nucleic acid analyte” or “analyte” is meant a nucleic acid sought tobe detected in a sample or a nucleic acid synthesized as a result of anucleic acid amplification reaction which contains at least about 20nucleotides of the nucleotide base sequence of a nucleic acid sought tobe detected in a sample or the complement thereof.

By “synthesizing” a nucleic acid or oligonucleotide is meant making thenucleic acid by chemical synthesis or enzymatic means. It is known thatcertain nucleic acid polymerase enzymes can incorporate modifiednucleotides during enzymatic synthesis.

By “modified”, a “modified nucleotide” or “modification” is meant apurposeful variant from the classical ribo- and deoxyribonucleotides A,T, G, C and U. When used in this specification, modified will mean avariant of the classical nucleotides, said variants leading to a higherbinding efficiency when an oligonucleotide which contains said modifiednucleotides is hybridized to a target nucleic acid than when the sameoligonucleotide contains the classical nucleotides. In some cases anoligonucleotide having a modified 3′ end may be referred to. This meansthat the 3′ end of the oligonucleotide contains a substitution whichinhibits or prevents extension of the 3′ end by a nucleic acidpolymerase.

By “conjugate molecule” is meant a molecule that can couple with anoligonucleotide in such a way that at least some of the characteristicsof both the molecule and the oligonucleotide are retained in thecombined product. Most often, the conjugate molecule contributes a newphysical or chemical property to the oligonucleotide, while theoligonucleotide retains its ability to base pair.

By “binding affinity” is meant a measure of the strength of hydrogenbonding between at least partly complementary nucleic acids underdefined nucleic acid hybridization conditions. A convenient measure ofbinding efficiency is the T_(m), which is the temperature at which 50%of said two strands are in the double-stranded or hybridized form.

By “label” is meant a reporter moiety which is capable of being detectedas an indication of the presence of the oligonucleotide to which it isjoined. When the labeled oligonucleotide is hybridized to one or moreother oligonucleotides, the presence of the label can be an indicationof the presence of the other oligonucleotide or oligonucleotides aswell. Appropriate reporter moieties are well known in the art andinclude, for example, radioisotopes, dyes, chemiluminescent,fluorescent, chemiluminescent and electrochemiluminescent compounds,nucleic acid sequences, enzymes, enzyme substrates, chromophores andhaptens.

By “nucleic acid assay conditions” is meant environmental conditions,including temperature and salt concentration, for the preferentialformation of stable hybrids between complementary base sequence regionsover the formation of stable hybrids between non-complementary basesequence regions.

By “acridinium ester derivative” or “AE” is meant any of a family ofchemiluminescent compounds derived from the acridinium ring which have alabelled ester or ester-like linkage at the C9 position connecting theacridinium ring to a leaving group. The leaving group is preferably anaryl or substituted aryl group. Substitutions, such as alkyl (e.g.,methyl), alkoxy (e.g., methoxy), aryl and halide (e.g., Br and F), maybe made to either or both the acridinium ring or the leaving group.Examples of such acridinium esters are provided in FIG. 1.

The methods and compositions of the present invention result from theunexpected discovery that oligonucleotides containing one or morenucleotides modified so that the oligonucleotides have an increasedT_(m) for a given target (as compared to otherwise identical unmodifiedoligonucleotides) will hybridize to a given target at an increased rateas compared to unmodified oligonucleotides. A maximum increase in thehybridization rate of a modified oligonucleotide occurs when a “cluster”of nucleotides are modified. By “cluster” is meant that at least about 4of 5 consecutive nucleotides are so modified. Thus, oligonucleotidescontaining a mixture of modified and unmodified nucleotides may be justas effective in increasing target hybridization rate as inoligonucleotides containing 100% modified nucleotides. Aspects of theinvention feature “chimeric” oligonucleotides containing both modifiedand unmodified nucleotides.

When used in this context, a “target” nucleic acid is a nucleic acidsought to be hybridized with an oligonucleotide. Such a nucleic acid maybe a naturally occurring nucleic acid, e.g., ribosomal RNA, it may bethe product (i.e., an “amplicon”) of nucleic acid amplification methodssuch as PCR or a transcription-based amplification method, as describedmore fully below, or it may be another synthetic oligonucleotide.

Thus, the present invention is directed to diagnostic methods andcompositions involving modified oligonucleotides which display anincrease in the rate of oligo:target hybridization over an unmodifiedoligonucleotide of the same base sequence.

In a preferred embodiment, the invention utilizes modifications to the2′ position of the deoxyribofuranosyl (or ribofuranosyl) ring. The2′-modification involves the placement of a group other than hydrogen orhydroxyl at the 2′ position of the ribofuranosyl ring. Regardless of thenature of the substitution, it must not sterically hinder the ability ofan oligonucleotide containing one or more such nucleotide modificationsto hybridize to a single-stranded oligonucleotide having a complementarynucleotide base sequence. The hybridization of complementary,double-stranded nucleic acids in which one strand contains suchmodifications is markedly increased as compared to situations in whichneither strand is so modified.

When a modified oligonucleotide is referred to as having an “increased”or “greater” affinity or rate, it is meant that the rate ofhybridization or affinity of the modified oligonucleotide is greaterthan the hybridization rate or binding affinity of an unmodifiedoligonucleotide of the same length and base sequence to the same target.

Particularly preferred oligonucleotides are substituted with a methoxygroup at the 2′ sugar position. These 2′-modified oligonucleotidesdisplay a preference for RNA over DNA targets hating a sequenceidentical to the RNA target (but having T substituted for U), withrespect to both T_(m) and hybridization kinetics. Other sucholigonucleotides are known in the art.

Conjugate molecules attached to oligonucleotides modified as describedherein may function to further increase the binding affinity andhybridization rate of these oligonucleotides to a target. Such conjugatemolecules may include, by way of example, cationic amines, intercalatingdyes, antibiotics, proteins, peptide fragments, and metal ion complexes.Common cationic amines include, for example, spermine and spermidine,i.e., polyamines. Intercalating dyes known in the art include, forexample, ethidium bromide, acridines and proflavine. Antibiotics whichcan bind to nucleic acids include, for example, actinomycin andnetropsin. Proteins capable of binding to nucleic acids include, forexample, restriction enzymes, transcription factors, and DNA and RNAmodifying enzymes. Peptide fragments capable of binding to nucleic acidsmay contain, for example, a SPKK (serine-proline-lysine(arginine)-lysine (arginine)) motiff, a KH motiff or a RGG(arginine-glycine-glycine) box motiff. See, e.g., Suzuki, EMBOJ,8:797-804 (1989); and Bund, et al., Science, 265:615-621 (1994). Metalion complexes which bind nucleic acids include, for example, cobalthexamine and 1,10-phenanthroline-copper. Oligonucleotides represent yetanother kind of conjugate molecule when, for example, the resultinghybrid includes three or more nucleic acids. An example of such a hybridwould be a triplex comprised of a target nucleic acid, anoligonucleotide probe hybridized to the target, and an oligonucleotideconjugate molecule hybridized to the probe. Conjugate molecules may bindto oligonucleotides by a variety of means, including, but not limitedto, intercalation, groove interaction, electrostatic binding, andhydrogen bonding. Those skilled in the art will appreciate otherconjugate molecules that can be attached to the modifiedoligonucleotides of the present invention. See, e.g., Goodchild,Bioconjugate Chemistry, 1(3):165-187 (1990). Moreover, a conjugatemolecule can be bound or joined to a nucleotide or nucleotides eitherbefore or after synthesis of the oligonucleotide containing thenucleotide or nucleotides.

Applicant has also unexpectedly discovered that the observed increase inhybridization rate of modified oligonucleotides to their targets doesnot always increase indefinitely with an increasing number of contiguousmodified nucleotides, especially when the target possesses an open orunfolded structure, or when helper probes are present. Under suchcircumstances, placement of modified nucleotides in a substantiallycontiguously arrangement, i.e., about 4 of 5 contiguous nucleotides, inthe oligonucleotide, followed by hybridization to a complementarytarget, will result in an increased hybridization rate only up to agiven number of modifications. The addition of modified nucleotides tothe cluster past this number will not, in general, substantially furtherincrease the hybridization rate.

In a currently preferred modification, employing 2′-O-methyl-substitutednucleotides, optimal hybridization rates are obtained inoligonucleotides having a cluster of about 8 contiguous modifiedresidues. Given the discovery that modified oligonucleotides canincrease the hybridization rate, it was unexpected that this effect doesnot always parallel the increase in T_(m) contributed by such additions.That is, addition of modified oligonucleotides past the rate-optimalcluster size will continue to increase the T_(m).

Although Applicant does not wish to be limited by theory, it is believedthat such clusters function as “nucleation centers” which are the firstregions of the oligonucleotide or nucleic acid to hydrogen-bond, in arate-limiting step, followed by rapid hydrogen bonding of the remainingbases. While the entire oligonucleotide or nucleic acid may be somodified, little advantage in increased hybridization rate appears to begained by substantially exceeding the optimal cluster size.

However, when the structure of the target is closed or folded in nature,and no helper probes are included, the hybridization rate between theoligonucleotide and the target generally may be improved by addingmodified nucleotides to the oligonucleotide in excess of about 4contiguous nucleotides. In a preferred embodiment, substantially all ofthe nucleotides of an oligonucleotide complementary to the structurallyclosed target will be modified.

The rate of in-solution hybridization of two complementarysingle-stranded nucleic acids depends on various factors, such as theconcentration of the nucleic acids, the temperature of hybridization,and the properties of the solvent solution, such as salt concentration.Various methodologies have been employed to increase hybridizationrates, the majority of which involve either changing the solvent system,such as by forming emulsions of immiscible solvents; by employingnucleic acid precipitating agents (e.g., Kohne et al., U.S. Pat. No.5,132,207), or volume excusion agents, such as polyethylene glycol; orby increasing the concentration of a nucleic acid strand.

A problem associated with the latter approach in a diagnostic assay isthat the target nucleic acid is usually present in quite small amounts.Thus, to increase the nucleic acid concentration necessitates using anexcess of the oligonucleotide, resulting in increased cost and reagentwaste and, if the oligonucleotide is labeled, risking unacceptably highbackgrounds. The other methods, such as those requiring the use ofmultiple solvents and agents, such as polyethylene glycol, may presentpractical difficulties, such as excessive sample manipulation and time.

Therefore, one aspect of the present invention provides a means forincreasing hybridization rates, as well as binding affinity, ofoligonucleotides for RNA targets by using oligonucleotides containingnucleotides having a substitution at the 2′ position of theribofuranosyl ring (“2′-modified oligonucleotide”); preferably an alkoxysubstitution, most preferably a methoxy substitution. These propertiesrender useful methods employing such oligonucleotides in a diagnostichybridization assay format by increasing the rate and extent ofhybridization of such an oligonucleotide without requiring a concomitantincrease in the concentrations of the hybridizing nucleic acids, achange in the properties or composition of the hybridization solution,the addition of “helper oligonucleotides”, disclosed in Hogan &Milliman, U.S. Pat. No. 5,030,557, or an increase in the hybridizationtemperature. U.S. Pat. No. 5,030,557 enjoys common ownership with thepresent invention and is incorporated by reference herein. Nonetheless,the methods of the present invention may be used as supplements to oneor more of these other techniques in any procedure in which an increasein the rate of nucleic acid hybridization would be advantageous.

As mentioned above, an advantage of this aspect of the invention relatesto the ability of 2′-O-methyl modified oligonucleotides topreferentially hybridize to RNA over DNA. This property allows thedesign of oligonucleotide probes targeted to RNA. Probes can be madewhich would not tend to bind to DNA under stringent hybridizationconditions, even where the DNA sequence is identical to the RNA targetsequence (except that T is substituted for U in the DNA sequence). Suchproperties can be used in a number of different formats in which thespecific detection of RNA would be advantageous. For example, toindicate and measure changes in the rate of transcription of particularRNA species, such as, for example, specific mRNA species, to monitor theeffectiveness of a given therapy, to specifically probe tRNA or rRNA inpreference to the genes encoding these RNA species, and to specificallydetect RNA viruses in nucleic acid preparations containing large amountsof chromosomal DNA, even DNA preparations containing DNA versions of theviral sequences.

An additional advantage of this and other aspects is an increasedtarget:oligo T_(m) when using modified oligonucleotides, such as2′-modified nucleotides as compared to the T_(m) of target:oligo hybridsin which the oligonucleotide is a deoxyoligonucleotide. By“target:oligo” is meant a hydrogen bonded, double-stranded nucleic acidcomplex comprising a single-stranded oligonucleotide. The stability ofthe target:probe complex increases with an increase in the number of2′-modified nucleotide residues contained in the probe. By contrast, theincrease in the hybridization rate appears to be optimal in nucleicacids having a cluster of about 8 2′-modified nucleotide residues anddoes not increase significantly upon the addition of consecutivemodified oligonucleotide residues above that number. Further, “chimeric”oligonucleotides having at least one such cluster of modifiednucleotides could be designed to have greater hybridization rateswithout necessarily significantly increasing the T_(m) of theoligonucleotide as a whole to its target.

An increased T_(m) may be exploited in any diagnostic procedure in whichthe added stability of a nucleic acid duplex is desired. For example,higher hybridization temperatures can be used to accelerate thehybridization rate. A higher T_(m) also permits the use of substantiallyshorter oligonucleotides than were heretofore practical, thus resultingin a savings in the costs associated with producing oligonucleotides forhybridization, as well as other advantages, as mentioned above.

In other aspects and embodiments of the present invention, chimericoligonucleotides may contain a modified portion designed to bind totarget nucleic acid and may also contain a deoxynucleotide portion whichis either directly or indirectly able to bind to a solid phase-boundoligonucleotide. By way of example, and not of limitation, such anoligonucleotide may be a target capture oligonucleotide designed to binda target nucleic acid (e.g., in solution) and link the bound targetnucleic acid to a derivatized solid phase matrix, such as a bead,microsphere, polymeric substance, such as agarose or dextran or to amagnetized particle. Derivatives linked to such a matrix may includeantibodies, ligands, or wholly or partially single-strandedoligonucleotides having a specific nucleotide sequence, such as ahomopolymeric tract, designed to bind the capture oligonucleotide or anintermediate oligonucleotide. The bound target can then be furtherhybridized with a probe (either RNA, DNA or modified) and the unboundprobe washed free of the immobilized target:probe complex beforedetecting the presence of the target nucleic acid.

It may be advantageous in certain instances to raise the temperature forhybridizing a modified oligonucleotide to its target. As describedabove, increasing the hybridization temperature also increases the rateof hybridization, so long as the hybridization temperature issufficiently below the T_(m) of the desired hybrid. The hybridizationmethods claimed herein, employing modified oligonucleotides having ahigher T_(m) than their unmodified counterparts, can be conducted athigher temperatures than would otherwise be used. In such a case, therate increase associated with the modification alone is furtherincreased by the raised temperature.

What follows are examples of embodiments of the invention, which shouldnot be understood as limiting the scope of the invention thereto. Thoseskilled in the art will easily comprehend additional embodiments basedon the disclosure contained in this specification. Additionalembodiments are also contained within the claims which conclude thisspecification.

Modified Nucleic Acid Hybridization Assay Probes

Nucleic acid hybridization assays utilize one or more nucleic acidprobes targeted to, i.e., having a nucleotide base sequencesubstantially complementary to, a nucleic acid sought to be detected.Often the probe will comprise an oligonucleotide (a single-strandednucleic acid of between about 10 and about 100 nucleotides in length)which is synthetically made to have a given nucleotide sequence. By“substantially complementary” is meant that the oligo will bind to itstarget under appropriately selective conditions to form a hydrogenbonded duplex.

While a hybridization assay probe will generally be joined to adetectable label, the probe can be unlabeled and probe:target hybridsdetected by, for example, UV absorbance, HPLC chromatography, gelelectrophoresis and subsequent staining of the nucleic acid hybrid orother methods well known in the art. In hybridization assays, the probeand target are contacted with each other under conditions permittingstable and specific hybridization. The resulting hybrid is thenseparated from unlabeled hybrid and the label detected, or the label canbe detected under conditions allowing the detection of the hybrid inpreference to the unlabeled probe.

Methods for separating nucleic acids, such as gel exclusionchromatography, reverse phase chromatography, and hydroxyapatiteadsorption, are known in the art. Applicant prefers an assay formatwherein labeled hybrid and labeled unhybridized probe can be chemicallydifferentiated by virtue of the formation of a double helix. Aparticularly preferred assay format is the hybridization protectionassay (HPA), see U.S. Pat. No. 5,283,174 to Arnold et al., in whichlabeled unhybridized probe can be selectively be made undetectable whilehybridized probe is relatively unaffected. Thus, detection of label inthis format is an indication of the labeled hybrid.

In these aspects, the present invention involves the use of modifiedoligonucleotides as probes in a hybridization assay. In one embodiment,modified oligonucleotides having a higher target-specific T_(m) thanunmodified oligonucleotides of the same base sequence are used toincrease the rate of hybridization of the assay, as compared to assaysemploying unmodified oligonucleotides of the same base sequence. Suchassay methods are able to utilize higher hybridization temperatures thanare practicable using unmodified oligonucleotides. The higherhybridization temperature further increases the rate of hybridizationand can also reduce the amount of cross-hybridization (hybridization ofthe probe with non-target sequences), thereby increasing the specificityof the assay.

The probes of this invention may comprise a cluster of about 4 or moresubstantially contiguous, modified nucleotide residues mixed withunmodified residues. Alternatively, the probe may comprise 100% modifiedresidues. In preferred embodiments, the modifications are 2′substitutions, such as alkyl, alkoxy and halide substitutions to the 2′carbon of the ribofuranosyl nucleotide moiety. In particularly preferredembodiments, the substitution is a methoxy group.

Particular probe modifications, including 2′-O-methyl substitutions,result in the oligonucleotide having an increased affinity and increasedhybridization rate to RNA targets but little effect on DNA affinity orrate of formation of probe:DNA hybrids. Again, this preference to RNAhas been found to be optimized when the oligonucleotide has at least onecluster of from about 4 to about 8 modified bases.

Because oligonucleotides modified as described herein have adramatically increased rate of hybridization, such modifiedoligonucleotide probes may be used in many cases without the need forthe addition of unlabeled “helper probes”, which are disclosed in Hogan,supra, as a means for increasing hybridization rates of probe to target.Nevertheless, Applicant has found that in some cases the combined use ofsuch modified oligonucleotides and helper probes may operate in concertto increase the hybridization rate even further. In either case, the useof such modified oligonucleotides in diagnostic methods leads to morerapid identification of biological analytes, which in turn leads to, forexample, to more effective treatments for disease conditions caused byor indicated by such analytes.

As described in more detail below, Applicant has found that probesdisplaying a preferential affinity to RNA targets may be used tospecifically detect RNA over DNA of the same sequence (except that U issubstituted for T in the RNA sequence). Such methods have applicationin, for example, the specific detection of RNA viruses in cellscontaining DNA versions of the viral genome, or in specific detection oflevels of RNA transcription in cells.

Probes may also be devised which contain both target-complementarysequences as well as additional target, non-complementary sequences.These target, non-complementary sequences may have other functions. Forexample, the sequences may be complementary to another oligonucleotideor target nucleic acid, or they may have functional properties, such aspromoter sequences and restriction sites. Thus, a probe may have morethan one function, only one of which is to be detected as an indicationof the presence of a target.

Additionally, probes may be designed to have at least one nucleic acidstrand which has at least two separate target-complementary sequencesthat can hybridize to a target nucleic acid. An example of such probesis described by Hogan et al., U.S. Pat. Nos. 5,424,413 and 5,451,503,which enjoy common ownership with the present application and areexpressly incorporated by reference herein. The probes disclosed byHogan et al. further include at least two distinct arm regions that donot hybridize with the target, but possess complementary regions thatare capable of hybridizing with one another. These arm regions can bedesigned to require the presence of target in order for theircomplementary sequences to hybridize under suitable hybridizationconditions. Accordingly, target-complementary sequences of the probemust hybridize to the target before complementary arm regions canhybridize to each other. The resulting structure is termed a branchednucleic acid.

Other probes may be designed which are unable to specifically bind thetarget. To be useful in detecting target, these probes must be able tohybridize with another nucleic acid that can bind with the target,either directly or indirectly. In one such arrangement, a nucleic acidcan be structured to contain at least a first and second, nonoverlappingnucleotide base sequence regions, where the first nucleotide region iscomplementary to a nucleotide base sequence of the target and the secondnucleotide region is complementary to a nucleotide base sequence of theprobe. In this arrangement, the second nucleotide region of the nucleicacid would be unavailable for binding with the probe until the targethas hybridized with the first nucleotide region of the nucleic acid.Binding of the nucleic acid and target would alter the configuration ofthe nucleic acid, thus permitting the second nucleotide region of thenucleic acid to bind with the probe. See FIG. 2. Of course, thisarrangement could be modified so that indirect binding between thenucleic acid and target, accomplished with one or more intervening orcoupling nucleic acids, would render the second nucleotide region of thenucleic acid available for binding with the probe.

Modified Nucleic Acid Amplification Oligonucleotides

In still other aspects, the present invention includes methods foremploying modified oligonucleotide primers, promoter-primers, and/orsplice templates for nucleic acid amplification and compositionscomprising such oligonucleotides, wherein the oligonucleotides containat least one cluster of modified bases which cause an increased rate ofhybridization.

Primer-employing amplification methods include the polymerase chainreaction method (PCR) and its variations, as described by Mullis, etal., (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, EuropeanPatent Application Nos. 86302298.4, 86302299.2, and 87300203.4, and 155Methods in Enzymology, 335-350 (1987)). The PCR methodology is by now amatter of common knowledge to those skilled in the art.

PCR has been coupled to RNA transcription by incorporating a promotersequence into one of the primers used in the PCR reaction and then,after amplification by the PCR method, using the double-stranded DNA asa template for the transcription of single-stranded RNA. (See, e.g.Murakawa, et al., DNA, 7:287-295 (1988)).

Other amplification methods use multiple cycles of RNA-directed DNAsynthesis and transcription to amplify DNA or RNA targets. See, e.g.,Burg, et al., U.S. Pat. No. 5,437,990; 89/1050; Gingeras, et al., WO88/10315; Davey and Malek, EPO Publication No. 0 329 822; Malek, et al.,WO91/02818, Kacian and Fultz, U.S. Pat. No. 5,480,783; McDonough, etal., WO 94/03472; and Kacian, et al., WO 93/22461 (the latter three ofthese publications enjoy common ownership with the present applicationand are incorporated by reference herein). Urdea, et al., WO91/10746,describe a method that achieves signal amplification using a T7 promotersequence.

Each of these methods makes use of one or more oligonucleotide primersor splice templates able to hybridize to or near a given nucleotidesequence of interest. After hybridization of the primer, thetarget-complementary nucleic acid strand is enzymatically synthesized,either by extension of the 3′ end of the primer or by transcription,using a promoter-primer or a splice template. In some amplificationmethods, such as PCR, rounds of primer extension by a nucleic acidpolymerizing enzyme is alternated with thermal denaturation ofcomplementary nucleic acid strands. Other methods, such as those ofKacian & Fultz, supra, McDonough, et al., supra, and Kacian, et al.,supra, are isothermal transcription-based amplification methods.

In each amplification method, however, side reactions caused byhybridization of the primer to non-target sequences can reduce thesensitivity of the target-specific reaction. These competing“mismatches” may be reduced by raising the temperature of the reaction.However, raising the temperature may also lower the amount oftarget-specific primer binding as well.

Thus, according to this aspect of the invention, primers having hightarget affinity, and comprising modified nucleotides in the targetbinding region, may be used in nucleic acid amplification methods tomore sensitively detect and amplify small amounts of a target nucleicacid sequence, by virtue of the increased temperature, and thus theincreased rate of hybridization to target molecules, while reducing thedegree of competing side-reactions (cross-reactivity) due tonon-specific primer binding. Preferred oligonucleotides contain at leastone cluster of modified bases, but less than all nucleotides aremodified in preferred oligonucleotides.

In another preferred embodiment, modified oligonucleotide primers areused in a nucleic acid amplification reaction in which a target nucleicacid is RNA. See, e.g., Kacian and Fultz, supra. The target may be theinitially present nucleic acid in the sample, or may be an intermediatein the nucleic acid amplification reaction. In this embodiment, the useof preferred 2′-modified primers, such as oligonucleotides containing2′-O-methyl nucleotides, permits their use at a higher hybridizationtemperature due to the relatively higher T_(m) conferred to the hybrid,as compared to the deoxyoligonucleotide of the same sequence. Also, dueto the preference of such 2′-modified oligonucleotides for RNA over DNA,competition for primer molecules by non-target DNA sequences in a testsample may also be reduced. Further, in applications wherein specificRNA sequences are sought to be detected amid a population of DNAmolecules having the same (assuming U and T to be equivalent) nucleicacid sequence, the use of modified oligonucleotide primers havingkinetic and equilibrium preferences for RNA permits the specificamplification of RNA over DNA in a sample.

Sample Processing

In accord with the present invention, modified oligonucleotides havingincreased target-specific hybridization kinetics and binding affinitiesas compared to their unmodified analogues may be used in a variety ofhybridization assay sample processing methodologies.

By sample processing is meant methods allowing or enhancing thediscrimination of analyte and non-analyte nucleic acids. Such methodsmay involve, for example, the direct or indirect immobilization ofnucleic acids or oligonucleotides from the liquid phase in aheterogeneous assay. Some such methods may involve two or morehybridization events resulting in such immobilization.

For example, Ranki, et al., U.S. Pat. Nos. 4,486,539 and 4,563,419,discuss a one-step nucleic acid “sandwich” hybridization methodinvolving the use of a solid-phase bound nucleic acid having a targetcomplementary sequence and a labeled nucleic acid probe, complementaryto a separate portion of the target nucleic acid. Stabinsky, U.S. Pat.No. 4,751,177, discusses methods involving the use of a “mediator”polynucleotide which reportedly overcomes sensitivity problems in theRanki method associated with leakage of the immobilized oligonucleotidefrom the solid support.

Other methods may employ an immobilized oligonucleotide, for example anoligonucleotide containing a homopolymeric tract, such as poly T, or asimple short repeating sequence, and two or more couplingoligonucleotides, one of which is able to hybridize with the immobilizedoligonucleotide and a different one of which is able to specificallyhybridize with target. Each of the coupling oligonucleotides is able tobind at least one other coupling oligonucleotide. If a couplingoligonucleotide does not contain a sequence complementary to the targetor immobilized oligonucleotide, it will be able to hybridize with atleast two other coupling oligonucleotides simultaneously. The solidsupport may be comprised of materials including nitrocellulose, apolymeric substance, such as polyacrylamide or dextran, metallicsubstances or controlled pore glass. The support may be in forms such asa sheet, membrane or a particle. Additionally, the solid support mayhave a magnetic charge to facilitate recovering sample and/or washingaway unbound nucleic acids or other sample components.

Joining of the immobilized oligonucleotide to the solid support may beaccomplished by any method that will continue to bind the immobilizedoligonucleotide throughout the assay steps. Additionally, it isimportant that when the solid support is to be used in an assay, it beessentially incapable, under assay conditions, of the non-specificbinding or adsorption of non-target oligonucleotides or nucleic acids.

Common immobilization methods include binding the nucleic acid oroligonucleotide to nitrocellulose, derivatized cellulose or nylon andsimilar materials. The latter two of these materials form covalentinteractions with the immobilized oligonucleotide, while the formerbinds the oligo through hydrophobic interactions. When using thesematerials it is important to use a “blocking” solution, such as thosecontaining a protein, such as bovine serum albumin (BSA), or “carrier”nucleic acid, such as salmon sperm DNA, to occupy remaining availablebinding sites on the solid support before use in the assay.

Other immobilization methods may include the use of a linker arm, forexample, N-hydroxysuccinamide (NHS) and its derivatives, to join theoligonucleotide to the solid support. Common solid supports in suchmethods are, without limitation, silica, polyacrylamide derivatives andmetallic substances. In such a method, one end of the linker may containa reactive group (such as an amide group) which forms a covalent bondwith the solid support, while the other end of the linker containsanother reactive group which can bond with the oligonucleotide to beimmobilized. In a particularly preferred embodiment, the oligonucleotidewill form a bond with the linker at its 3′ end. The linker is preferablysubstantially a straight-chain hydrocarbon which positions theimmobilized oligonucleotide at some distance from the surface of thesolid support. However, non-covalent linkages, such as chelation orantigen-antibody complexes, may be used to join the oligonucleotide tothe solid support.

A desirable embodiment of the latter assay system contains two couplingoligonucleotides: (i) a first coupling oligonucleotide containing anucleotide sequence substantially complementary to the immobilizedoligonucleotide, for example, with a poly A nucleotide sequencecomplementary to a poly T sequence on the immobilized oligo, and (ii) asecond coupling oligonucleotide containing a nucleotide sequencesubstantially complementary to the target nucleic acid, a detectablylabeled probe, or both. In a preferred embodiment, the second couplingoligonucleotide contains a nucleotide sequence substantiallycomplementary to the target nucleic acid. Moreover, each couplingoligonucleotide in the preferred embodiment contains another nucleotidesequence which enables the first and second coupling oligonucleotides tohybridize to each other under assay conditions. However, one or moreadditional coupling oligonucleotides may be introduced into the system,such that the first and second coupling oligonucleotides are indirectlybound to each other by means of these additional, intervening couplingoligonucleotides. The additional coupling oligonucleotides would besubstantially unable to hybridize with the any of the target nucleicacid, the detectably labeled oligonucleotide probe, or the immobilizedoligonucleotide under assay conditions.

Yet another assay system having practical advantages in ease andrapidity of use may comprise an immobilized oligonucleotide having aportion complementary to a capturing oligonucleotide. The capturingoligonucleotide (capture probe) will contain a base sequence permittinghybridization to the target. The capturing oligonucleotide will alsohave a label attached within or near the target-binding nucleotidesequence region, such as a substituted or unsubstituted acridiniumester, which may be used in a homogeneous or semi-homogenous assaysystem to specifically detect hybrid nucleic acids without detectingsingle-stranded nucleic acids, such as the capture probe itself. Such asystem favored by Applicant is the ™ HPA, which is discussed andincorporated by reference above. In the ™ HPA format, the labelcontained on any capture probe which has not hybridized to its targetwill be hydrolyzed with the addition of base, while target:capture probehybrid would protect the label associated therewith from hydrolysis.

An advantage to this latter assay system is that only onetarget-specific hybridization event (labeled capture probe:target) needoccur for target detection, rather than two such events (captureprobe:target and labeled probe:target) in the other sample processingprocedures described herein. Fewer oligonucleotides in the assay wouldtend to make the assay faster and simpler to optimize, since the overallrate at which labeled target is captured is limited by the slowesthybridizing probe. Additionally, while the portion of the targetcomplementary to the capturing oligonucleotide in these other assaysystems does not have to be as specific as the target's probe bindingregion, this base sequence must be rare enough to avoid significantsaturation of the capture probe with non-target nucleic acids. Thus,this preference for two separate and specific target sequences may placeconstraints on finding an appropriate target to which such assays are tobe directed. By contrast, only one such target sequence need be found inthe latter assay, since the same nucleotide sequence functionssimultaneously to immobilize and detect the target nucleic acid.

Regardless of the approach used, a necessary element of any assay is amethod of detection of the desired target. A number of options known tothose of skill in the art are possible. One such option is the directuse of a labeled nucleic acid probe. Such a probe would have anucleotide sequence region which is specifically hybridizable with andsubstantially complementary to the target nucleic acid of interest. Uponhybridization to the target and immobilization of the target:probehybrid, unbound probe can be washed away or inactivated and theremaining label hybrid-associated detected and/or measured.

Another option combines the elements of detection and nucleic acidamplification. In such a system, the target nucleic acid is immobilizedas described, for example, and without limitation, in the assayprocedures described above. One or more amplification oligonucleotides(see, e.g., Kacian, et al., WO93/22461), such as a primer,promoter-primer, or splice template, able to hybridize with a specificregion of the target nucleic acid may be contacted with the immobilizedtarget nucleic acid under nucleic acid amplification conditions, e.g, inthe presence of one or more nucleic acid polymerases and ribo- and/ordeoxyribonucleotide triphosphates.

The resulting polynucleotide strand (amplicon) can be made directlyavailable for specific hybridization and detection with a labeledhybridization assay probe or for further amplification by hybridizingthe polynucleotide strand with one or more additional amplificationoligonucleotides under nucleic acid amplification conditions. If thelatter option is chosen, the amplification reaction can be continueduntil the desired level of amplification is achieved, then the resultingamplicons, which may comprise copies of at least a portion of theimmobilized target nucleic acid, polynucleotides complementary to atleast a portion of the immobilized nucleic acid, or both, can bedetected using one or more labeled oligonucleotide probes. If theamplification reaction is to take place while the target is immobilized,it is important that the portion of the target molecule to be used as atemplate for the amplicons not contain the nucleotide sequence regionnecessary for immobilization of the target nucleic acid. Althoughamplicons of either or both senses can be detected with the labeledprobes, in a preferred embodiment only amplicons of the opposite senseto, i.e., complementary to, the immobilized target are detected.

A heterogeneous target capture method such as this is particularlyadvantageous since crude clinical samples can contain substances whichinhibit or interfere with the amplification reaction. Thus, the abilityto separate the target nucleic acid from such interfering substances canpermit or enhance the sensitivity of nucleic acid amplification.

This solid-phase associated amplification scheme can be used in myriadassay systems, including those described above. Applicant currentlyprefers an assay system employing one or more coupling oligonucleotides,as described above, which are able to indirectly link the target nucleicacid to the solid support. It is also preferred that the complementarynucleotide sequence regions of the support-coupled oligonucleotide, andthe capturing oligonucleotide designed to hybridize to it, be at leastpartially homopolymeric or contain simple repeating nucleotidesequences, so as to promote rapid hybridization. Additionally, oralternatively, these regions of either or both the immobilizedoligonucleotide and the capturing oligonucleotide may be modified in amanner consistent with the disclosure of this specification to increasethe rate of hybridization between these oligonucleotides. In such asystem, the target capture oligonucleotide and the target nucleic acidsare preferably allowed to hybridize in solution before hybridizing tothe immobilized oligonucleotide. In the currently preferred embodiment,the immobilized target is washed, the amplification oligonucleotide (oroligonucleotides) is contacted with the immobilized target under nucleicacid amplification conditions, and following amplification the labeledamplicon-directed probe is added and detected.

Applicant prefers to use the transcription-based amplification methoddescribed in Kacian & Fultz, supra, previously incorporated byreference. In accord with this method, a promoter-primer having a 3′region complementary to a portion of the target and a 5′ region and aprimer having the same nucleotide sequence as a portion of the targetare contacted with a target RNA molecule. The primer and promoter-primerdefine the boundaries of the target region to be amplified, includingboth the sense present on the target molecule and its complement, andthus the length and sequence of the amplicon. In this preferredembodiment, the amplification oligonucleotides and immobilized targetRNA are contacted in the presence of effective amounts of Moloney murineleukemia virus-derived reverse transcriptase and T7 RNA polymerase, bothribonucleotide and deoxyribonucleotide triphosphates, and necessarysalts and cofactors at 42° C. Under these conditions, nucleic acidamplification occurs, resulting predominantly in production of RNAamplicons of a sense opposite to that of the target nucleic acid. Theseamplicons are then detected, e.g., by using an acridinium ester-labeledhybridization assay probe of the same sense as the target-nucleic acid,in the hybridization protection assay disclosed in Arnold, supra,previously incorporated by reference.

In this preferred embodiment, Applicant prefers that the 3′ terminus ofthe immobilized oligonucleotide, target capture oligonucleotide andcoupling oligonucleotide(s) be “capped” or blocked to prevent or inhibittheir use as templates for nucleic acid polymerase activity. Capping mayinvolve addition of 3′ deoxyribonucleotides (such as cordycepin), 3′,2′-dideoxynucleotide residues, non-nucleotide linkers, such as disclosedin Arnold, et al., supra, alkane-diol modifications, ornon-complementary nucleotide residues at the 3′ terminus.

Although Applicant currently prefers to contact the primers with thetarget following target immobilization, there may be hybridizationkinetic advantages to combining the target nucleic acid and at least oneprimer complementary thereto at the same time that the target captureoligonucleotide is added. Applicant believes that it is advantageous toconduct the target hybridization in solution prior to immobilization ofthe target, as hybridization can take place in solution more rapidlythan when one nucleic acid is immobilized.

Likewise, while Applicant prefers to form and detect amplicons of theopposite sense to the target, there is no reason why one could not formand detect amplicons of either or both senses. Additionally, whenamplifying target nucleic acids contained in crude clinical samples, itappears important to conduct a wash step prior to the amplifying step toprevent enzyme inhibition and/or nucleic acid degradation due tosubstances present in the sample.

It will be clear to the skilled person that this methodology isamenable, either as described or with obvious modifications, to variousother amplification schemes including the polymerase chain reaction.

Modified Oligonucleotides in Sample Processing

Modified oligonucleotides able to hybridize to complementary targetswith increased kinetics may be used in sample processing methods whichemploy nucleic acid hybridization, including the target capture methodsdescribed above. In light of the present disclosure it will be apparentthat such partly or wholly modified oligonucleotides may be employed ashybridization assay probes or amplification oligonucleotides in thesesystems. Additionally, wholly or partially modified oligonucleotideshaving increased target-directed hybridization kinetics may be used asimmobilized oligonucleotides, target capture oligonucleotides, and/orone or more coupling oligonucleotides in heterogenous assays employingnucleic acid hybridization. For example, such modifications may be usedto reduce the overall assay time or to allow the hybridization steps ofthe assay to occur at a single temperature. The advantages of reducedtime and ease of assay operation in a clinical setting gained therebywould be clear to those skilled in the art.

Additionally, oligonucleotides may be modified to have hybridizationkinetics and/or equilibrium preferences for a specific type of nucleicacid, such as RNA or DNA. As disclosed above, for example, 2′-O-methyloligonucleotides preferentially hybridize with RNA over DNA. Thus,target capture oligonucleotides containing 2′-O-methyl nucleotides maybe used to specifically capture RNA target nucleic acids, such as mRNAor rRNA, under hybridization conditions not promoting hybridization ofthe oligonucleotide to the genomic versions thereof. Likewise,2′-O-methyl-modified amplification oligonucleotides and/or labeledprobes can be designed, thereby targeting RNA over DNA for amplificationand/or detection, as described above.

Modified Helper Oligonucleotides

Helper oligonucleotides are described in the Hogan, supra. Helperoligonucleotides are generally unlabeled and used in conjunction withlabeled hybridization assay probes to increase the labeled probe's T_(m)and hybridization rate by “opening up” target nucleotide sequenceregions which may be involved in secondary structure, thus making theseregions available for hybridization with the labeled probe.

In light of the present disclosure, those of skill in the art willeasily recognize that using modified helper oligonucleotides which willhybridize with the target nucleic acid at an increased rate over theirunmodified counterparts can lead to even greater hybridization rates ofthe labeled probe to their target. Thus, methods and compositions fordetecting oligonucleotides employing such modified helperoligonucleotides are intended to be encompassed within the scope of thisinvention. Preferred helper oligonucleotides have modifications whichgive them a greater avidity towards RNA than DNA. In a preferredembodiment, such modifications include a cluster of at least about 42′-O-methyl nucleotides. In a particularly preferred embodiment, suchmodifications would include a cluster of about 8 2′-O-methylnucleotides.

Diagnostic Kits

The methods described herein also clearly suggest diagnostic kitsspecially formulated for use in such methods. These kits will containone or more oligonucleotides to be used in a diagnostic nucleic acidhybridization assay. At least one of these oligonucleotides will containa cluster of at least about 4 modified nucleotides designed to hybridizeto a target nucleic acid region at an increased rate over an otherwiseidentical oligonucleotide.

Such diagnostic kits may include, without limitation, one or anycombination of the probe, amplification, helper and sample processingoligonucleotides described herein.

In a preferred embodiment of the present invention, the kit contains atleast one labeled oligonucleotide probe having a region containing oneor more clusters of at least about 4 contiguous 2′-modified nucleotideresidues. In a more preferred embodiment, the region contains one ormore clusters of about 8 2′-modified nucleotides.

Applicant currently prefers using an acridinium ester derivative as anon-radioactive label and the addition of a methoxy group as a 2′modification. In a particularly preferred embodiment, at least one ofthe modified oligonucleotides will comprise one or more clusters of atleast about 4 2′-O-methyl nucleotides. Even more preferred is at leastone oligonucleotide containing one or more clusters of about 8 2′-Omethyl nucleotides.

Kits containing one or more of the modified oligonucleotides disclosedherein could be sold for use in any diagnostic hybridization assaymethod, or related amplification method, of the present invention. Insuch an assay, at least one of the modified oligonucleotides containedin the kit would function as a probe able to hybridize to a targetnucleic acid. If the modified probe is contacted with a samplecontaining the target nucleic acid, the probe will exhibit improvedhybridization properties over an unmodified probe having an identicalbase sequence. For instance, the hybridization binding affinity betweenthe target and the probe will be greater than the hybridization bindingaffinity between the target and an unmodified form of the probe, whensubjected to the same hybridization assay conditions. Additionally, thehybridization rate between the target and the probe will be greater thanthe hybridization rate between the target and an unmodified form of theprobe, when subjected to the same hybridization assay conditions.

To further improve the hybridization properties of the probe, one ormore conjugate molecules may bound to the probe, preferably in a regioncontaining a cluster of at least about 4 modified nucleotides. It isalso expected that the kit would be packaged with instructions for usingone or more modified oligonucleotides in a diagnostic hybridizationassay of the present invention.

Objects

It is therefore an object of the present invention to provide methodsfor increasing the both the avidity of binding and the hybridizationrate between a diagnostic nucleic acid probe and its target nucleic acidby utilizing probe molecules having one or more modified nucleotides,preferably a cluster of about 4 or more, and more preferably about 8,modified nucleotides. In preferred embodiments, the modificationscomprise 2′ modifications to the ribofuranosyl ring. In most preferredembodiments the modifications comprise a 2′-O-methyl substitution.

It is also an object to provide methods for increasing the rate ofhybridization of a single-stranded oligonucleotide to a target nucleicacid through the incorporation of a plurality of modified nucleotidesinto the oligonucleotide. An increased rate of hybridizationaccomplished in this manner would occur over and above the increase inhybridization kinetics accomplished by raising the temperature, saltconcentration and/or the concentration of the nucleic acid reactants.

It is another object of the invention to provide diagnostic methods forselectively targeting RNA over DNA through the use of oligonucleotidesmodified to have an increased target binding efficiency and to hybridizeto RNA at an enhanced rate over DNA. In a preferred embodiment, sucholigonucleotides comprise a 2′-O-methyl modification to theribofuranosyl ring.

It is an additional object of the invention to provide sample processingmethods which employ an immobilized oligonucleotide to directly orindirectly capture target nucleic acids. In preferred embodiments, suchmethods employ one or more oligonucleotides which can specificallyhybridize to the target nucleic acid, permitting its detection andimmobilization. In a preferred embodiment, a single labeledoligonucleotide is responsible for both capture and detection of thetarget. In a particularly preferred embodiment, a coupling or bridgingnucleic acid is bound to both the immobilized oligonucleotide and theoligonucleotide responsible for capture and detection of the target.Additional coupling nucleic acids are possible. Some or all of theoligonucleotides used in sample processing methods may containmodifications which accelerate the rate of target specifichybridization.

It is yet another object of the invention to provide target specificoligonucleotides of between about 10 and about 100 bases, preferablybetween about 10 and about 16 bases, and more preferably between about12 and about 16 bases, which preferably contain at least one cluster ofat least about 4 nucleotides, more preferably about 8 nucleotides,modified to increase their target-specific binding efficiency whilesimultaneously increasing their discrimination between target andnon-target nucleotide sequences as compared to longer unmodifiedoligonucleotides designed to hybridize to the same site.

It is a further object of the present invention to provide kitsincluding one or more oligonucleotides containing modified nucleotideswhich function to increase the rate of hybridization between theoligonucleotide and a target nucleic acid. Kits of the present inventioncould include any combination of probe, amplification, helper and sampleprocessing oligonucleotides. In a preferred embodiment, the modifiedoligonucleotides of these kits would contain at least one cluster ofabout 4 2′-O-methyl modifications to the ribofuranosyl ring. Kitscontaining these modified oligonucleotides may be supplied for use inboth diagnostic hybridization assays and amplification assays. Such kitsmay further include written instructions directing practitioners in theuse of the modified oligonucleotides in either or both diagnostichybridization assays or amplification assays.

The diagnostic methods of the present invention are, therefore,specially adapted to exploit the hybridization properties of modifiedoligonucleotides having increased binding affinity. These methods may beused for the detection or quantification of any target nucleic acid. Ina preferred embodiment, the target nucleic acid is RNA. The methods mayemploy “chimeric” oligonucleotides composed of regions of unmodifiedoligodeoxy- or oligoribonucleotides combined with regions of modifiedoligonucleotides or may utilize wholly modified oligonucleotides.Preferably, the oligonucleotides are not wholly modified. The regionsmay be designed simply to promote rapid hybridization of probe totarget, or may have other functions. For example, a chimericoligonucleotide may be designed to bind both to RNA and to DNA. In sucha case, the RNA-binding portion of the oligonucleotide may contain aplurality of modified nucleotides to preferentially bind the RNA target.Alternatively, the region of modified residues may be designed to bedirected towards a target present in low abundance in order to increasethe hybridization rate.

Given the present disclosure, it will be understood that certainembodiments of the methods and compositions, including the kits, of thepresent invention may employ oligonucleotides having more than one typeof modification affecting the hybridization properties of the resultingoligonucleotide, i.e., T_(m) and hybridization kinetics. Such multiplemodifications may act in a cooperative fashion to further increase thehybridization rate or to increase the specificity of the resultingoligonucleotide for a given type of nucleic acid target, such as RNA.Furthermore, chimeric oligonucleotides may have or consist of regions ofdifferently modified oligonucleotides containing either 2′-modifiednucleotides or nucleotides having other modifications or both.

The objects and aspects of the invention specifically described hereinare not intended as an exhaustive listing of the objects or aspects ofthe methods and compositions of the present invention which would beapparent to those skilled in the art in light of the present disclosure.Nor should the preceding description or the Examples which follow beconstrued as limiting the invention to the embodiments specificallydisclosed therein.

EXAMPLES

Unless otherwise indicated, in all the following examplesoligodeoxyribonucleotides, oligoribonucleotides, and modifiedoligonucleotides were synthesized by use of standard phosphoroamiditechemistry, various methods of which are well known in the art. See e.g.,Carruthers, et al., 154 Methods in Enzymology, 287 (1987), which ishereby incorporated by reference as part of this disclosure. Unlessotherwise stated herein, modified nucleotides were 2′O-methyl-nucleotides, which were used in the synthesis as theirphosphoramidite analogs. Applicant prepared the oligonucleotides usingan Expedite 8909 DNA Synthesizer (PerSeptive Biosystems, Framingham,Mass.).

Also, unless otherwise indicated, oligonucleotides indicated as labeledcontained an acridinium phenyl ester. Acridinium phenyl ester compoundsare derivatives of acridine possessing a quaternary nitrogen center andderivatized at the 9 position to yield a phenyl ester moiety. However,leaving groups other than phenyl moieties are well known in the art.Acridinium esters have the property of reacting with hydrogen peroxideto form a transient dioxetane ring involving the C-9 carbon of theacridinium ring, followed by the formation of an excited acridone. Theradiative relaxation of the excited acridone results in the productionof light. The synthesis of acridinium esters, as well as a generaldescription of their use as chemiluminescent labeling reagents, isdescribed in Weeks, et al., Acridinium Esters as High Specific ActivityLabels in Immunoassays, Clin. Chem., 29:1474-1478 (1984), which isincorporated by reference herein.

In these Examples, the acridinium esters were attached, using standardchemical techniques, to a non-nucleotide monomeric unit having a primaryamine “linker arm” joined to the acridinium ester moiety, which isinserted between contiguous sequences of nucleotides during the chemicalsynthesis of the oligonucleotides, or placed at a terminal position ofthe oligonucleotide. See, Arnold, et al., Non-Nucleotide LinkingReagents for Nucleotide Probes, EPO Publication No. EPO 313219, whichenjoys common ownership with the present invention, and is nowincorporated by reference herein. However, it will be understood thatthe preference of 2′-modified oligonucleotides for RNA targets and theeffect of the modified oligonucleotides on the rate of hybridization toDNA targets are not determined by the presence or specific nature of alabel. Thus, those of skill in the art will recognize thatoligonucleotides used in the methods of the present invention may belabeled with a variety of labels, or they may be unlabelled when, forexample, they are used as amplification primers, helper oligonucleotidesor in a capture assay.

Acridinium ester derivatives may be joined to the linkerarm:hybridization probe conjugate using techniques well known in theart. Preferably, Applicant uses the methods described in Nelson, et al.,Detection of Acridinium Esters by Chemiluminescence in Non-IsotopicProbe Techniques (Academic Press 1992), and Arnold, et al.,Non-Nucleotide Linking Reagents for Nucleotide Probes, EPO PublicationNo. EPO 313219, previously incorporated by reference herein.

Further, unless expressly indicated otherwise, all target nucleic acidswere RNA.

It will nevertheless be clear to those of skill in the art, in light ofthe present disclosure, that other labels may be used in the methods andcompositions of the present invention without departing from the spiritof the invention disclosed herein.

Example 1 Effect of 2′ Modifications on the T_(m) of Probe:TargetHybrids

Oligonucleotide probes of identical sequence containing varying amountsof 2′-O-methyl nucleotides were each individually hybridized toperfectly complementary synthetic RNA targets of the same length. Thetarget sequence (SEQ ID NO: 1) and the probe sequences (SEQ ID NO:2-6)were as follows (reading 5′ to 3′):

SEQ ID NO:1: atgttgggttaagtcccgcaacgagc; SEQ ID NO:2:gctcgttgcgggacttaacccaacat (Probe A); SEQ ID NO:3:gcucguugcgggacuuaacccaacau (Probe B); SEQ ID NO:4:gcucguugcgggacttaacccaacau (Probe C); SEQ ID NO:5:gctcgttgcgggaciiaacccaacat (Probe D); and SEQ ID NO:6:gctcgttgcgggacuuaacccaacat (Probe E).

These probes, were synthesized to contain no 2′-O-methyl nucleotides(Probe A), all 2′-O-methyl nucleotides (Probe B), or a combination ofdeoxy- and 2′-O-methyl nucleotides (Probes C, D and E), and each probewas labeled with an acridinium phenyl ester compound joined to a linkerarm attached to the probe between nucleotides 16 and 17 (reading 5′ to3′). The bolded nucleotides represent 2′-O-methyl nucleotides. Probe Ccontained four contiguous deoxyribonucleotides positioned directlyadjacent to each side of the linker attachment site and 2′-O-methylribonucleotides at all other bases; Probe D contained four contiguous2′-O-methyl nucleotides positioned directly adjacent to each side of thelinker attachment site and deoxyribonucleotides at all other bases, andProbe E contained eight contiguous 2′-O-methyl nucleotides positioneddirectly adjacent to each side of the linker attachment site anddeoxyribonucleotides at all other bases. The T_(m) of each hybrid wasdetermined using both a chemiluminescent and an optical method.

Chemiluminescent Method

Using the chemiluminescent method, approximately 1 pmol of the RNAtarget and 0.1 pmol of each oligonucleotide probe, labeled as describedabove with “standard” acridinium ester (4-(2-succinimidyloxycarbonylethyl) phenyl-10-methylacridinium 9-carboxylate fluorosulfonate) wereallowed to hybridize at 60° C. for 60 minutes in 30 μl of lithiumsuccinate buffer (1.5 mM EDTA (ethylenediaminetetraacetic acid), 1.5 mMEGTA (ethylene glycol-bis (β-aminoethyl ether) N,N,N′,N′-tetraaceticacid), 310 mM lithium lauryl sulfate, 0.1 M lithium succinate (pH 5.2)).The resulting solution was then diluted to 500 μl with lithium succinatebuffer, and 50 μl aliquots were incubated at various temperatures for 7minutes. Each sample was then cooled on ice for an additional 7 minutes.The acridinium ester coupled to unhybridized probe molecules washydrolyzed by adding 150 μl of a solution containing 190 mM Na₂B₄O₇ (pH7.6), 7% (v/v) TRITON® X-100 (polyoxyethylene p-t-octyl phenol) and0.02% (w/v) gelatin, and the samples were heated at 60° C. for 10minutes. The remaining (hybrid associated) chemiluminescence of eachsample was determined in a LEADER® 50 luminometer (MGM Instruments;Hamden, Conn.) by the automatic injection of a solution containing 0.1%v/v H₂O₂ in 0.001 M HNO₃ followed 0.5-2 seconds later by an injection of200 μl of 1N NaOH. The resulting light emission was integrated over a 2second interval.

Optical Method

Using the optical method, an identical set of oligonucleotide probeswere synthesized having a linker arm but were not labeled with anacridinium ester. Four micrograms of each oligonucleotide probe wereallowed to hybridize to 4 μg of the complementary RNA target for 60minutes at 60° C. in 30 μl of a hybridization buffer containing 200 mMlithium hydroxide, 3 mM EDTA, 3 mM EGTA, 17% w/v lithium lauryl sulfate,and 190 mM succinic acid (pH 5.2). Following hybridization, 600 μl ofthe hybridization buffer was added, the sample split into two, and themelting behavior of each sample portion examined on a Beckman DU640spectrophotometer equipped with a Micro T_(m) analysis accessory. Thetemperature was varied 1° C. per minute for temperatures that were morethan 10° C. either lower or higher than the T_(m) and 0.5° C. per minuteat intervals of 0.2° C. for all other temperatures. Changes inhypochromaticity were monitored and recorded as a function oftemperature. Results are shown in Table 1 below.

TABLE 1 Number of modified Probe nucleotides T_(m) (chemiluminescent)T_(m) (optical) Δ T_(m) A  0 68 72.4  0 B 26 90 91.2 22; 18.8 C 18 8387.9 15; 15.5 D  8 72 76.4  4; 4 E 16 nd 84.2 11.6 nd = not done

As shown in Table 1, the T_(m) data generated using the chemiluminescentand optical methods agreed well with each other. The somewhat lowerT_(m) values observed with the chemiluminescent method can be attributedto the lower nucleic acid concentrations used in the chemiluminescentmethod versus the optical method. The data show that replacement of allof the deoxyribonucleotide residues of Probe A with 2′-O-methylnucleotides (Probe B) resulted in probe:RNA target hybrids having aT_(m) increased by about 20.4° C. Probes C, D and E exhibited T_(m)increases of 15° C., 4° C., and 11.6° C., respectively. By calculatingthe effect on T_(m) for each substitution of a 2′-O-methyl nucleotide,these data reveal that the T_(m) of the 2′-O-methyl oligonucleotide:RNAtarget hybrid increases about 0.8° C. for every such replacement. Thiseffect is approximately linear over the number of substitutions tested.

Example 2 Effect of 2′-Modified Nucleotides on T_(m) of Probe:rRNAHybrids

Three sets of oligonucleotide probes of different length and sequencewere synthesized, and each set contained two oligonucleotides ofidentical base sequence. Probe F was 17 bases in length and included anacridinium ester label joined at a site located between a thymine baseand an adenine base. Probe G was 18 bases in length and likewiseincluded an acridinium ester label joined at a site located between athymine base and an adenine base. Probe H was 20 bases in length andincluded an acridinium ester label joined at a site located between athymine base and a guanine base.

Each set of probes contained one oligonucleotide consisting entirely ofdeoxyribonucleotides and another oligonucleotide containing only2′-O-methyl nucleotides. Each probe was then hybridized to thecorresponding ribosomal RNA, and the T_(m) of the resulting hybridsdetermined by the chemiluminescent method described above. The resultsare shown in Table 2 below.

TABLE 2 Δ T_(m) per T_(m) (2′-O- modified Probe Length (bases) T_(m)(deoxy) methyl) Δ T_(m) nucleotide F 17 63 81 18 1.05 G 18 66 78 12 0.66H 20 62 75 13 0.65

The data confirm the results of Example 1, showing that replacement of adeoxyribonucleotide with a 2′-O-methyl nucleotide increases the T_(m) ofthe resulting probe:RNA target hybrid. Additionally, when calculated asthe average of the three probes' increase in T_(m) per modifiednucleotide, the contribution of each modified nucleotide was an increaseof 0.8° C. per modified nucleotide.

Example 3 Effect of 2′-Modified Nucleotides on T_(m) of Probe:DNAHybrids

In this Example, the effect of 2′-modification on probe:DNA targets wastested. Probe I, which contained varying amounts of 2′-O-methylnucleotides, was hybridized to an exactly complementary DNA target ofthe same length and the melting behavior of the resultant hybridsexamined by the chemiluminescent method described above. Probe I was 29bases in length and included an acridinium ester label joined at a sitelocated between a thymine base and a guanine base.

Probe I was designed to consist of: (i) all deoxyribonucleotides; (ii)all 2′-O-methyl nucleotides; and (iii) all 2′-O-methyl nucleotidesexcept for four deoxyribonucleotides, which were positioned immediatelyon each side of the label attachment site. Results of the T_(m)determination are shown in Table 3 below.

TABLE 3 Number of 2′-O-methyl Δ T_(m) per modified Probe nucleotidesT_(m) nucleotide I  0 69 0 I 29 77 0.28 I 21 75 0.29

As the data shows, replacement of deoxyribonucleotides with 2′-O-methylnucleotides in Probes J and K caused the T_(m) of the labeled probe:DNAtarget to increase approximately 0.3° C. per 2′-O-methyl residue.

A similar test was done using three sets of different oligonucleotides.Each set contained two oligonucleotides, one of the oligonucleotidescontaining deoxyribonucleotides and the other containing 100%2′-O-methyl nucleotides, having identical base sequences. Probe J was 16bases in length and included an acridinium ester label joined at a sitelocated between a thymine base and an adenine base. Probe K was 18 basesin length and likewise included an acridinium ester label joined at asite located between a thymine base and an adenine base. Probe L was 29bases in length and included an acridinium ester label joined at a sitelocated between a thymine base and a guanine base.

In each case the synthetic DNA targets were completely complementary tothe probes. The results are shown in Table 4 below.

TABLE 4 Number of 2′-O-methyl T_(m) Δ T_(m) per modified Probenucleotides (optical method) nucleotide J  0 75.3 0 J 26 74.5 −0.03 K  071.6 0 K 19 67.0 −0.24 L  0 74 0 L 29 78.2 0.14

The data contained in Tables 3 and 4 demonstrates that the T_(m) of DNAtargets is increased to a significantly lesser degree than RNA targetswhen 2′-O-methyl substitutions are introduced into the probes.

Example 4 Analysis of the Stabilities of Different Types of Nucleic AcidHybrids

To compare the relative stabilities of hybrids containing variouscombinations of DNA, RNA, and 2′-O-methyl nucleotide strands, acridiniumester-labeled oligonucleotide probes were hybridized to synthetictargets having a perfectly complementary base sequences (reading 5′ to3′):

SEQ ID NO:2: gctcgttgcgggacttaacccaacat (DNA); SEQ ID NO:3:gcucguugcgggacuuaacccaacau              (2′-O-methyl nucleotides); andSEQ ID NO:7: gcucguugcgggacuuaacccaacau (RNA).As in Example 1, each probe was labeled with an acridinium phenyl estercompound joined to a linker arm attached to the probe betweennucleotides 16 and 17 (reading 5′ to 3′). The base sequences of thetarget sequences were as follows (reading 5′ to 3′):

SEQ ID NO:1: atgttgggttaagtcccgcaacgagc (DNA); SEQ ID NO:8:auguuggguuaagucccgcaacgagc              (2′-O-methyl nucleotides); andSEQ ID NO:9: auguuggguuaagucccgcaacgagc (RNA).The probes and target sequences contained 100% (RNA) (SEQ ID Nos. 7 and9), 100% (DNA)(SEQ ID Nos. 1 and 2) or 100% 2′-O-methyl nucleotides (SEQID Nos. 3 and 8) in the combinations indicated in Table 5. The meltingcharacteristics of each tested hybrid, as determined either using thechemiluminescent or the optical method, is shown in Table 5 below. Morethan one data point in the table indicates an independent, duplicateexperiment.

TABLE 5 T_(m) Probe Target (chemiluminescent) T_(m) (optical) DNA RNA68, 67 73.3, 73.6, 73, 72.4 RNA RNA 81 nd 2′-O-methyl RNA 87, 90 91.2nucleotides 2′-O-methyl 2′-O-methyl 91 nd nucleotides nucleotides DNADNA nd 75.5, 75.7, 75.1, 75.4 2′-O-methyl DNA nd 74.3, 74.8 nucleotidesnd = no data

Thus, this experiment indicates that the stability of labeledprobe:target hybrids follows the order:2′-O-methyl/2′-O-methyl≧2′-O-methyl/RNA>RNA/RNA>DNA/DNA>2′-O-methyl/DNA>DNA/RNA

Example 5 Ability of Enhanced Stability of 2′-Modified Oligo:RNA Hybridsto Allow Specific RNA Targeting

As indicated in Example 4, 2′-O-methyl:RNA hybrids are considerably morestable than 2′-O-methyl:DNA hybrids. To illustrate that this differencein stability can be exploited in a diagnostic assay to specificallydetect RNA molecules over DNA molecules having an identical sequence(but with uracil in the RNA replacing thymine in the DNA ), thefollowing experiment was done.

The acridinium ester-labeled oligonucleotide probe of SEQ ID NO:3 (seeExample 1 above) was allowed to hybridize to a completely complementarysynthetic RNA target (SEQ ID NO:9) or DNA target (SEQ ID NO:1). Otherthan the fact that the oligonucleotide was labeled, hybridization andmeasurement of T_(m) were otherwise as described in Example 1 under theheading Optical Method. The results are shown in Table 5 above and arefurther illustrated in FIG. 3.

As indicated in FIG. 3, at 81.6° C. the 2′-O-methyl oligonucleotideforms a detectable hybrid with the RNA target, but not with the DNAtarget. By contrast, Table 5 demonstrates that when a labeled DNAoligonucleotide of the same sequence is hybridized with the identicalRNA or DNA targets, the resulting hybrids have substantially similarmelting characteristics.

Thus, according to the aspect of the present invention demonstratedhere, it is possible to specifically detect RNA targets in preference toDNA targets under easily determined hybridization conditions. Suchmethods may be used, as a non-exclusive example, to specifically detectvarious RNA species, such as mRNA, tRNA, or rRNA, without interferencefrom the identical sequence existing in the genomic DNA of the organismbeing assayed. Such methods may be useful for applications includingmonitoring the rate of expression of a particular gene product. Otheruses exploiting this ability of the 2′-modified oligonucleotides will beapparent to those of skill in the art.

Example 6 Effect of 2′-Modified Nucleotides on the HybridizationKinetics of Oligonucleotides

The effect of 2′-modified nucleotides on hybridization kinetics wasillustrated using four different methods. The probe molecules used inthis example were labeled with standard acridinium ester as describedpreviously.

a) In the first approach, 2 fmol of an acridinium ester-labeled probehaving the sequence of SEQ ID NO:2 or SEQ ID NO:3 (see Example 1 above)was hybridized to varying amounts of a completely complementary RNAtarget (SEQ ID NO:9) for a constant period of time, followed bydifferential hydrolysis and detection of the label. The hybridizationwas performed essentially as described in Example 1, under the headingChemiluminescent Method, with the following differences. Varying amountsof RNA target were allowed to hybridize with the labeled probe at 60° C.for 45 minutes. FIG. 4 shows the results of this experiment, wherein theprobe had either the DNA sequence of SEQ ID NO:2 (open boxes) or the2′-O-methyl nucleotide sequence of SEQ ID NO:3 (closed diamond); theseresults are also tabulated in Table 6 below. The degree of hybridizationis expressed in Relative Light Units (rlu), which is a measure of thenumber of photons emitted by the acridinium ester label.

TABLE 6 Amount of Target (fmol) rlu (DNA probe) rlu (2′-O-methyl)  1  855  1,739  5  3,394  8,009 10  5,476 14,217 50 13,810 27,959 100 18,798 34,381 200  19,199 31,318

The results indicate that the hybridization rate, as a function oftarget concentration, is significantly increased when the probe contains2′-O-methyl nucleotides rather than unmodified nucleotides. This holdstrue throughout the range of target concentrations studied. Forcomparison purposes, the initial slopes of these data are used toestimate the relative hybridization rates of deoxy- (slope=1.0) and2′-O-methyl (slope=2.5) oligonucleotide probes.

b) In a second approach, a constant amount (2 fmol) of the same targetused in a) above was hybridized to varying amounts of the perfectlycomplementary probe for a fixed amount of time. FIG. 5 shows the resultsof this experiment, wherein the probe had either the DNA sequence of SEQID NO:2 (open boxes) or the 2′-O-methyl nucleotide sequence of SEQ IDNO:3 (closed boxes). The hybridization and detection steps were the sameas described in a) above, except that the hybridization reaction wascarried out for 30 minutes rather than 45 minutes. The data aretabulated in Table 7 below.

TABLE 7 Amount of Probe (fmoles) rlu (DNA probe) rlu (2′-O-methyl) 0.2  99   346 0.5   309   966 1   690  1,973 2 1,206  4,356 5 3,227  9,18410 5,801 14,615 20 7,289 21,515 50 13,080  33,236 100 15,223  34,930

Again, the results in this example indicate that the hybridization rate,which is a function of probe concentration, is significantly increasedwhen the probe contains 2′-O-methyl nucleotides rather than unmodifiednucleotides. The slopes of these plots are similar to those of FIG. 4,indicating that regardless of whether probe concentration or targetconcentration is varied, the difference in hybridization kineticsbetween 2′-O-methyl/DNA and DNA/DNA interactions remains the same. Inthis experiment, the initial slope of the reaction containing the DNAprobe was 1.0 and of the reaction containing the 2′-O-methyl probe was3.1.

c) As a third illustration of the ability of 2′-modifiedoligonucleotides to increase the rate of hybridization, fixed amounts ofeither modified or unmodified probe (1 fmol) and target (100 amol) wereallowed to hybridize for varying amounts of time. The hybridization anddetection protocols were otherwise the same as in b). FIG. 6 shows theresults, wherein the probe had either the DNA sequence of SEQ ID NO:2(open boxes) or the 2′-O-methyl nucleotide sequence of SEQ ID NO:3(closed diamonds). The data were as follows in Table 8 below:

TABLE 8 Time (minutes) rlu (DNA probe) rlu (2′-O-methyl) 0 118 613 1 124627 2 200 732 6.5 294 978 10.5 500 1331 

Again, the relative rates of hybridization can be determined from theinitial slopes of the curves (deoxy=1.0; 2′-O-methyl=2.2). In thisexperiment, the initial slope of the reaction containing the DNAoligonucleotide was 1.0, and the initial slope of the reactioncontaining the 2′-O-methyl oligonucleotide probe was 2.2-fold.

d) The fourth method used to demonstrate the differences between thehybridization kinetics of 2′-modified and unmodified probes was a C_(o)tanalysis. Acridinium ester-labeled probes of SEQ ID Nos. 2 and 3 (seeExample 1 above) were used. Either a fixed amount of probe and variedamounts of target (“probe excess”) or a fixed amount of target andvarying amounts of probe (“target excess”) were allowed to hybridize at60° C. for varying amounts of time. The fixed amount of either probe ortarget was 0.25 fmol and the variable amount of either probe or targetincluded amounts in the range from 0.25 to 50 fmol. Hybridization wasotherwise as indicated in Example 1, under the heading ChemiluminescentMethod.

Differential hydrolysis of the unhybridized acridinium ester anddetection of the hybridized probe was accomplished by adding 150 μl of190 mM Na₂B₄O₇ (pH 7.6), 7% (v/v) TRITON® X-100 and 0.02% (w/v) gelatinto the sample, and heating the mixture to 60° C. for 10 minutes.Chemiluminescence was read in a luminometer, as described above. Percentmaximal hybridization was defined as the ratio of the observed rlu valuedivided by the maximal rlu value observed when saturating amounts ofprobe or target were used in the hybridization reaction.

In the C_(o)t plot shown in FIG. 7, the quantity in (1−H) is plottedagainst the concentration of target times the hybridization time. Thevalue H is defined as the percent hybridization of the probe at aparticular target concentration after a particular time.

In this plot, the relative rates of hybridization of DNA and 2′-O-methyloligonucleotide probes is given by the inverse of the relative ratios ofC_(o)t at 50% hybridization (ln(1−0.5); deoxy=1.0 and 2′-O-methyl=2.2).

A summary of the relative hybridization rates of an acridiniumester-labeled probe consisting entirely of deoxy- or 2′-O-methylribonucleotides determined by these four methods is summarized in Table9. The data resulting from these experiments indicate that at 60° C. anoligonucleotide consisting entirely of 2′-O-methyl nucleotideshybridizes 2.3-fold faster than the corresponding deoxyribonucleotideprobe.

TABLE 9 Relative Rate Hybridization (2′-O-methyl/ Method Probe (fmol)Target (fmol) Time deoxy-) c 0.05 1 vary 2.3 c 10 0.5 vary 2.3 c 0.1 1vary 2.0 c 1 0.2 vary 1.6 c 3 0.3 vary 2.9 c 10 1 vary 1.8 b vary 1 302.6 b vary 2 45 3.1 a 2 vary 45 2.5 d 0.25 vary 44 2.2

Example 7 Kinetic Analysis of Hybridization of 2′-ModifiedOligonucleotides to Additional Targets

To extend these hybridization rate comparisons to other probe and targetsequences, Probe H of Example 2 above was synthesized entirely of eitherdeoxy- or 2′-O-methyl nucleotides and hybridized to rRNA in the presenceof helper probes. A C_(o)t analysis was performed, as in Example 6(d).The results are shown in Table 10 below.

TABLE 10 Probe C_(o)t_(1/2) Relative Rates deoxy 0.3 × 10⁷ 1.002′-O-methyl   8 × 10⁷ 3.75

In this Example, the probe consisting entirely of 2′-O-methylnucleotides hybridized 3.75-fold faster than the deoxyribonucleotideprobe of identical sequence. Thus, enhanced hybridization by acridiniumester-labeled probes containing 2′-O-methyl nucleotides does not appearto be limited to any particular probe or target sequence.

Example 8 Effect of Increased Temperature on Hybridization Rate of2′-Modified Oligonucleotides

As mentioned above, nucleic acid hybridization kinetics are acceleratedby an increase in temperature. However, the advantages of thisacceleration can be offset by the destabilizing effects of increasedtemperature on duplex formation, especially in diagnostic assays andnucleic acid amplification procedures employing relatively shortoligonucleotides (between about 10 and about 50 bases in length). Asshown below, the increased duplex stability provided by oligonucleotidesmodified as presently described can minimize this destabilizing effect,allowing the hybridization rate to be further increased by conductingthe hybridization at a higher temperature than would otherwise possible.Thus, a cooperative effect on hybridization kinetics is provided by themodified oligonucleotides and the higher hybridization temperature.

An acridinium ester-labeled oligonucleotide probe having the sequence ofSEQ ID NO:3 (see Example 1 above) was allowed to hybridize to aperfectly complementary RNA target of the same length as describedabove. The hybridization and C_(o)t protocol were as described inExample 6(d), except that hybridization temperatures were either 60° C.or 70° C. As shown by the data in Table 11 below, raising thetemperature of hybridization of the 2′-O-methyl oligonucleotide to itstarget from 60° C. or 70° C. caused the hybridization kinetics to beaccelerated 1.5 fold.

TABLE 11 Hybridization Temperature C_(o)t_(1/2) Relative HybridizationRates 60° C. 1.7 × 10⁻⁵ 1 70° C. 1.1 × 10⁻⁵ 1.5

Example 9 Effect of Increasing Salt Concentration on HybridizationKinetics of 2′-Modified Oligonucleotides

Hybridization kinetics are also accelerated by increases in saltconcentration. The following example illustrates the effect of variousconcentrations of salt, e.g., LiCl, on the hybridization kinetics of2′-O-methyl nucleotides. An acridinium ester-labeled probe having thesequence of SEQ ID NO:3 (see Example 1 above), was allowed to hybridize,and a C_(o)t analysis conducted, as described in Example 6(d) above, at80° C. to an exactly complementary RNA target of the same length.Hybridization was performed at two different concentrations of LiCl. Asshown in Table 12 below, increasing the salt concentration from 0.5 to1.0 M LiCl enhanced the hybridization kinetics 2.9 fold.

TABLE 12 LiCl Concentration C_(o)t_(1/2) Relative Rates 0.5 M 0.72 ×10⁻⁵ 1 1.0 M 0.25 × 10⁻⁵ 2.9

These results demonstrate that a two-fold increase in the saltconcentration in a hybridization reaction leads to a 2.9-fold increasein the hybridization kinetics of modified oligonucleotides.

Example 10 Combined Effect on Hybridization Kinetics of Increasing SaltConcentration and Temperature

To demonstrate the effect of simultaneously increasing the hybridizationtemperature and salt concentration on the hybridization kinetics of2′-modified oligonucleotides, the following reactions were performed. Anacridinium ester-labeled DNA oligonucleotide having the sequence of SEQID NO:2 and an acridinium ester-labeled oligonucleotide having thesequence of SEQ ID NO:3 (see Example 1 above), were each separatelyallowed to hybridize to an exactly complementary RNA target molecule ina C_(o)t analysis. Hybridization conditions were as described in Example6(d). Hybridization temperature and salt concentrations were asindicated in Table 13 below. The results were as follows:

TABLE 13 Hybridization LiCl Relative Probe Temperature ConcentrationC_(o)t_(1/2) Rates DNA 60° C. 0.5 M  1.9 × 10⁻⁵ 1 2′-O-methyl 80° C. 1.0M 0.22 × 10⁻⁵ 8.6

As the data indicate, at a hybridization temperature of 80° C. and in1.0M LiCl, the 2′-O-methyl oligonucleotide hybridized to its target atan 8.6-fold faster rate than did the corresponding DNA oligonucleotideat a temperature of 60° C. and salt concentration of 0.5M LiCl.

Example 11 Comparison of Hybridization Rates of RNA, DNA and 2′-ModifiedOligonucleotides Hybridizing to Complementary DNA and RNA Targets

The relative hybridization rates of labeled RNA, DNA and2′-O-methyl-containing oligonucleotides to completely complementary DNAand RNA targets were individually determined. Rate determination wasperformed as disclosed in either Example 6(c) or 6(d) above. The labeledoligonucleotides had the sequences of SEQ ID Nos. 2, 3 and 7 (seeExample 1 above). The results are summarized in Table 14 below:

TABLE 14 Probe Target Initial Slope C₀t_(½) T_(m) Relative Rates DNA DNA.0031, .0048 — 75.4 4.4 RNA DNA — — 74.5 — 2′-O Me DNA .0014, .0014 —74.6 1.6 DNA RNA .0009 — 73.3 1 RNA RNA —  1.1 × 10⁻⁵, 81 2.8 1.4 × 10⁻⁵2′-O Me RNA .004   .71 × 10⁻⁵, 91.2 4.4 .89 × 10⁻⁵ Experimentsrepresented in Rows 1 through 4 were done as disclosed in Example 6(c)using 3 fmol of labeled probe and 0.3 fmol of target. The experimentsrepresented in rows 5 and 6 were performed using the method described inExample 6(d). Where more than one result is indicated, each valuecorresponds to a different individual experiment.

The results of Table 14 demonstrate that substitution ofdeoxyribonucleotide residues with either 2′-OH residues (RNA) or2′-O-methyl residues enhances the affinity, as well as the hybridizationkinetics, of the probe to an RNA target. In contrast, substitution ofdeoxyribonucleotide residues with 2′-O-methyl residues does not enhancethe affinity or the hybridization kinetics of the probe to a DNA target.

The results presented in Table 14 reveal that substitution ofdeoxyribonucleotide residues with 2′-O-methyl residues enhances theaffinity and hybridization kinetics of a probe for an RNA target, butnot a DNA target. However, these experiments do not eliminate thepossibility that the acridinium ester label and/or the linker, by whichit is attached to the probe, may be responsible for these hybridizationrate characteristics. To show that the acridinium ester label and/orlinker do not appreciably affect hybridization rates, the followingexperiment was performed.

An RNA probe of SEQ ID NO:7 labeled with acridinium ester (see Example 1above) and containing a non-nucleotide linker by which the label wasattached to the probe was allowed to hybridize to an exactlycomplementary target which consisted entirely of either 2′-O-methyl ordeoxyribonucleotides. Hybridization and C_(o)t analysis was done asExample 6(d). The results are expressed in Table 15 below.

TABLE 15 Labeled Probe Target C_(o)t_(1/2) Relative Rates RNA DNA 6.2 ×10⁻⁵ 1 RNA 2′-O-methyl 2.3 × 10⁻⁵ 2.7

These data reveal that substitution of deoxyribonucleotides with2′-O-methyl residues enhances the hybridization kinetics of anoligonucleotide lacking an acridinium ester and/or linker. Thus, theincreased hybridization rate observed for 2′-O-methyl modified probes isnot due to the presence of a label or linker arm, but is an intrinsicproperty of the 2′-O-methyl-modified oligonucleotide.

Example 12 Comparative Effect of Helper Probes on the Hybridization ofProbes from DNA Probe Mixes and 2′-Modified Probe Mixes to an rRNATarget

As disclosed in Hogan, supra, the hybridization of some probes tonucleic acids, especially those having a significant amount of secondarystructure, is facilitated through the use of additional probes, termed“helper” probes. An example, though not the exclusive example, of anucleic acid species having a high degree of secondary structure isribosomal RNA (rRNA). Helper probes can help disrupt secondary structurewhich may mask the target region. The helper probe is usually targetedto a sequence region near, but preferably not overlapping with, theprobe target sequence. In practice, helper probes are generally notlabeled, and are usually used in a large molar excess. The effect ofusing helper probes on labeled probe hybridization is usually expressedas an increased T_(m) and hybridization rate for the labeledprobe:target hybrid.

The following experiments were performed to examine whether 2′-modifiedoligonucleotide probes have the same requirements for helper probes asdo DNA probes when directed to given target regions containing a largeamount of secondary structure. Two probe mixes were made. Each probe mixcontained Probes F, G and H of Example 2 above. The oligonucleotides ofone probe mix were made up of 100% 2′-O-methyl nucleotides, and theoligonucleotides of the other probe mix were composed entirely ofdeoxyribonucleotides. Where indicated, probe mixes included unlabeledDNA helper probes a, b, c, d, e and f having lengths of 33, 36, 41, 31,29 and 40 bases, respectively.

Each helper probe was directed to rRNA base sequences close to thetarget site of one of the labeled probes. The degree of hybridizationwas measured using the hybridization protection assay (HPA), asdescribed above. The results are reported in relative light units (rlu).

As shown in Table 16 below, in the absence of helper probes, DNA probemixes hybridized poorly to rRNA. In contrast, when probe mixes employing2′-O-methyl probes of identical sequence to the DNA probe mixes werehybridized to rRNA in the absence of helper probes, much higher levelsof hybridization were observed. Additionally, when helper probes wereused with both probe mixes, significantly greater hybridization of theprobes to their target occurred with the 2′-modified oligonucleotideprobe mixes. Because the hybridization of a DNA probe to an rRNA dependsstrongly on the presence of helper probes, while the hybridization of anidentical 2′-O-methyl probe does not, 2′-O-methyl probes can efficientlyhybridize to highly structured RNA molecules, such as ribosomal RNA,under conditions where DNA probes cannot.

TABLE 16 rRNA Concentration Probe Helpers (amol) rlus DNA no 100 14 DNAyes 100 3185 2′-O-methyl no 100 3116 2′-O-methyl yes 100 4332 DNA no1,000 68 DNA yes 1,000 24,912 2′-O-methyl no 1,000 19,934 2′-O-methylyes 1,000 33,584 DNA no 10,000 730 DNA yes 10,000 204,876 2′-O-methyl no10,000 148,386 2′-O-methyl yes 10,000 256,940

The data in Table 16 further indicate that helper probes are not neededto facilitate probe binding when 2′-modified oligonucleotides are used.However, even greater sensitivity than seen before can be achieved inassays employing both helper probes and 2′-modified probes.

Example 13 Comparative Effect of Helper Probes on the Hybridization of aSingle DNA Probe and a Single 2′-Modified Probe to an rRNA Target

The results of the experiments described in Example 12 were generatedusing three separate labeled probes, either with or without the presenceof the six helper oligonucleotides identified in Example 12. In thisexample, Probes F and H of Example 2, and consisting entirely ofdeoxyribo- or 2′-O-methyl nucleotides, were allowed to hybridize totarget rRNA in the presence or absence of the indicated helper probes.Helper probes a, b, c and d of Example 12 above were used. Table 17shows the hybridization characteristics of DNA and 2-O-methyloligonucleotides of Probe A. Table 18 shows the hybridizationcharacteristics of DNA and 2′-O-methyl oligonucleotides of Probe H. Eachlabeled probe was tested in the presence or absence of the varioushelper probes and helper probe combinations indicated.

TABLE 17 Target Concentration Probe (amol) Helper Probes rlus deoxy 500None 153 deoxy 500 a 5,600 deoxy 500 b 761 deoxy 500 a and b 9,3632′-O-methyl 500 None 14,537 2′-O-methyl 500 a 16,556 2′-O-methyl 500 b15,586 2′-O-methyl 500 a and b 16,868 deoxy 1,000 None 1,060 deoxy 1,000a 14,782 deoxy 1,000 b 316 deoxy 1,000 a and b 26,877 2′-O-methyl 1,000None 28,874 2′-O-methyl 1,000 a 23,201 2′-O-methyl 1,000 b 16,2692′-O-methyl 1,000 a and b 44,510

TABLE 18 Probe Helper RLUs deoxy c   870 deoxy d  9,176 deoxy c and d47,745 2′-O-methyl c 88,292 2′-O-methyl d 69,943 2′-O-methyl c and d98,663

Tables 17 and 18 demonstrate that the labeled DNA probes required helperoligonucleotides in order to effectively hybridize to their targetsunder the assay conditions. Additionally, Table 18 shows that the2′-modified oligonucleotides hybridized to their targets to a greaterdegree in the absence of helper probes than the DNA oligonucleotideshybridized to the same target in the presence of the added helpers.Finally, 2′-O-methyl oligonucleotides exhibited even greaterhybridization properties in the presence of helper oligonucleotides.

Example 14 Comparative Effect of Temperature on the HybridizationProperties of DNA Probes and 2′-Modified Probes with an rRNA Target inthe Presence and Absence of Helper Probes

The data presented in Example 13 indicates that 2′-O-methyloligonucleotides hybridize to a detectable extent to RNA targets, evenhighly folded structures like rRNA, in the absence of helper probes.Nevertheless, helper probes can accelerate the hybridization of2′-O-methyl oligonucleotides to highly structured RNA. To examine theeffect of helper probes more closely, deoxy- and 2′-O-methyloligonucleotide probes were hybridized to rRNA at different temperaturesin the presence or absence of helper probes. Table 19 represents studiesperformed using acridinium ester-labeled probes having a nucleotidesequence of Probe F of Example 2 above and helper probes c and d ofExample 12 above. Table 20 represents studies performed using acridiniumester-labeled probes having the sequence of SEQ ID NO:3 (see Example 1above) and helper probes g and h having 41 and 32 bases, respectively.

TABLE 19 Probe Helpers Temperature C_(o)t_(1/2) Relative Rate deoxy Yes60° C. 26.4 × 10⁻⁵ 1.3 2′-O-methyl No 60° C.   35 × 10⁻⁵ 1 2′-O-methylYes 60° C. 8.15 × 10⁻⁵ 4.3 2′-O-methyl No 75° C. 12.9 × 10⁻⁵ 2.72′-O-methyl Yes 75° C. 4.12 × 10⁻⁵ 8.5

TABLE 20 Probe Helpers Temperature C_(o)t_(1/2) Relative Rate2′-O-methyl No 60° C. 7.46 × 10⁻⁵ 1 2′-O-methyl Yes 60° C. 1.77 × 10⁻⁵4.2

These experiments demonstrate that at 60° and 75° C., helper probesenhanced the hybridization rates of the 2′-O-methyl probes to theirtargets 3.1-4.3 fold.

Example 15 Effect of 2′-Modified Nucleotides on the HydrolysisProperties of Acridinium Ester-Labeled Probes

As a further demonstration of the effect of modified oligonucleotides onthe performance characteristics of diagnostic probe molecules, a numberof additional experiments were performed. These experiments were basedon the Applicant's preferred detection method employing the HPAdetection assay. In accordance with one HPA format, a chemiluminescentacridinium ester is attached to a probe and the probe is hybridized toan analyte. Following hybridization, chemiluminescence associated withunhybridized probe is selectively destroyed by brief hydrolysis inborate buffer. Since probe:analyte molecules are not destroyed in thisprocess, the remaining chemiluminescence of hybridized probe is a directmeasure of the analyte present. In this application, those acridiniumester-labeled probes which hydrolyze faster when unhybridized than whenin a probe-analyte hybrid complex are preferred. Hydrolysis of probe andhybrid is pseudo first order and can be characterized by the value t½,which is the time, measured in minutes, required to hydrolyze 50% of theacridinium ester attached to either probe or hybrid. Thus, probes whichexhibit a large differential hydrolysis (DH) ratio(t½(hybrid)/t½(probe)) are highly desirable.

To examine the effect of modified oligonucleotides on the hydrolysisproperties of acridinium ester-labeled probes, four sets of probes wereconstructed, each set having a distinct nucleotide base sequence andeach member of a set having identical nucleotide base sequences. Eachset of probes contained one probe consisting entirely of unmodifiednucleotides and another probe consisting entirely of 2′-O-methylnucleotides. The probes used were Probes A and B of Example 1 above andProbes F, G and H of Example 2 above. DH ratios of each probe to anexactly complementary RNA target were determined as described above, forexample, in Example 1. As summarized in Table 21 below, unhybridizedprobes containing deoxy- or 2′-O-methyl nucleotides hydrolyzed at verysimilar rates.

TABLE 21 Probe Ribonucleotides t½ (Probe) t½ (Hybrid) DH A A deoxy .8149.1 60.3 B 2′-O-methyl .63 77.2 123.5 B F deoxy .36 20.79 7.74 F2′-O-methyl .89 75.25 4.55 C H deoxy .69 17.26 25 H 2′-O-methyl .76 44.758.8 D G deoxy .62 25.67 41.4 G 2′-O-methyl .81 23.55 29.7

In contrast, the modified probe:target hybrid-associated label for threeof the sequences was approximately 2-fold more resistant to hydrolysisthan in the otherwise identical unmodified probe:target hybrid. In onecase, in which the probe contained an ATAT sequence surrounding theacridinium ester linker, the DH ratio was decreased 1.4-fold for themodified probe:target hybrid-associated label.

Example 16 Effect of Position of 2′-Modified Nucleotides on theHydrolysis Properties of Acridinium Ester-Labeled Probe

To examine whether modified nucleotides must be close to the site oflabel attachment to enhance the DH behavior of acridinium ester, theacridinium ester-labeled probes containing clusters of 2′-O-methylnucleotides at different positions relative to the acridinium esterlinker site, were hybridized to a complementary RNA target. LabeledProbes A, B, C and D of Example 1 were used for this example.

As shown in Table 22 below, the measurements reveal that 4 contiguous2′-O-methyl nucleotides on either side of the acridinium ester linkersite are sufficient to enhance the DH behavior of an acridiniumester-labeled probe as much as a probe consisting entirely of2′-O-methyl nucleotides. More than one data point in the table indicatesindependent, duplicate experiments.

TABLE 22 Probe t½ (Probe) t½ (Hybrid) DH A .82, .8 48.7, 43.2  59.7, 54B .76, .6 90, 77.6 118.3, 129 C .74 49.8  67.3 D .44 81.4 185

Example 17 Effect of Temperature on the Hydrolysis Properties of2′-Modified Acridinium Ester-Labeled Probes

As mentioned above, because of their higher thermal stability, themodified oligonucleotides of the present invention are able to hybridizeto a target nucleic acid at a higher temperature than unmodifiedoligonucleotides. At such higher temperatures, the hybridization rate,as well as the rate of other reactions, would be expected to increase.Among such other reactions is the rate of hydrolysis of acridinium esterlabels. Because Applicant's preferred detection method employs theHybridization Protection Assay HPA, described and incorporated byreference above, the following experiment was performed to determinewhether benefits to the diagnostic assay conferred by an increase inhybridization rate would be offset by a decrease in the DH ratios ofhybrid-associated and unassociated acridinium ester labels at thishigher temperature.

An acridinium ester-labeled probe consisting entirely of 2′-O-methylnucleotides was allowed to hybridize to a complementary RNA target at60°, 70° and 80° C. Hybridization conditions were as otherwise asdescribed previously.

As summarized in Table 23 below, at 70° C. and 80° C. acridiniumester-labeled probes containing 2′-O-methyl nucleotides exhibited DHratios comparable to acridinium ester-labeled probes containingdeoxyribonucleotides at 60° C. Thus, elevated temperature may be used indiagnostic assays employing the methods and compositions of the presentinvention without a detectable decrease in assay sensitivity due todegradation of the label.

TABLE 23 Temp t½ t½ (° C.) Probe (Probe) (Hybrid) DH 60 deoxy .82 48.759.7 60 2′-O-methyl .76 90   118   70 2′-O-methyl .42 25.7 61.3 802′-O-methyl .25 10.1 40.8

Example 18 Effect of 2′-Modified Nucleotides on the HydrolysisProperties of Various Acridinium Ester-Labeled Probes

The foregoing experiments were conducted using standard acridinium esteras the detectable chemiluminescent label. To examine whether thedifferential hydrolysis behavior of labels other than standardacridinium ester is enhanced by T_(m)-enhancing modified nucleotides,probes containing either deoxyribonucleotides (SEQ ID NO:2) or2′-O-methyl nucleotides (SEQ ID NO:3) were labeled in exactly the samemanner and position (see Example 1 above) with standard acridiniumester, o-diBr acridinium ester, 2-Me acridinium ester,napthyl-acridinium ester, o-F acridinium ester,2,7-diisopropylacridinium ester, or mixture of 1- and 3-Me acridiniumester, and their DH behavior examined. See FIG. 1 for examples ofacridinium esters. As summarized in Table 24 below, the use of modifiednucleotide probes resulted in an increase in the DH ratio for all theacridinium ester derivatives tested by 1.1-6 fold.

TABLE 24 t½ t½ Label Probe (Probe) (Hybrid) DH Standard Acridinium esterdeoxy .81 49.1 60.3 Standard Acridinium ester 2′-O-methyl .63 77.2 123.5o-diBr-acridinium ester deoxy .94 23.44 24.94 o-diBr-acridinium ester2′-O-methyl 1.01 66.65 66 2-Me-acridinium ester deoxy .84 73.6 87.622-Me-acridinium ester 2′-O-methyl .78 101.8 130.5 Napthyl acridiniumester deoxy .72 14.45 20.07 Napthyl acridinium ester 2′-O-methyl .5753.4 93.68 o-F acridinium ester deoxy .93 53.3 57.3 o-F acridinium ester2′-O-methyl 1.03 78.75 76.46 2,7-diisopropyl deoxy 1.23 38.55 31.3acridinium ester 2,7-diisopropyl 2′-O-methyl 0.8 43.90 54.9 acridiniumester Mixture of 1- and 3-Me deoxy .97 12.6 129.8 acridinium esterMixture of 1- and 3-Me 2′-O-methyl 1.24 149.5 120.6 acridinium ester

Example 19 Relationship Between the Hybridization Kinetics and theNumber of 2′-Modified Nucleotides Contained in a Probe Sequence

To examine the relationship between hybridization kinetics and thenumber of 2′-O-methyl nucleotides within a probe sequence, Probes A, B,D and E of Example 1 were synthesized, as well as the following probesequences (reading 5′ to 3) labeled in the same manner and location asthe probe of Example 1 with an acridinium phenyl ester compound:

SEQ ID NO:10: gctcgttgcgggacttaacccaacat (Probe M); and SEQ ID NO:11:gctcgttgcgggacttaacccaacat (Probe N).The bolded nucleotides represent 2′-O-methyl nucleotides. The resultsare summarized in Table 25 below.

TABLE 25 2′-O-methyl Relative Residues C_(o)t (Exp 1) C_(o)t (Exp 2)C_(o)t (Exp 3) Rate  0 1.87 × 10⁻⁵  2.2 × 10⁻⁵ — 1    2 — — 1.13 × 10⁻⁵1.8  4 1.28 × 10⁻⁵ — 0.93 × 10⁻⁵ 1.8  8 — 0.93 × 10⁻⁵ 0.86 × 10⁻⁵ 2.3 16— — 0.96 × 10⁻⁵ 2.1 25 0.91 × 10⁻⁵   1 × 10⁻⁵ 0.84 × 10⁻⁵ 2.2

As summarized above, as few as 8 2′-O-methyl nucleotides (Probe D)—4 oneach side of the acridinium ester linker site—were sufficient toaccelerate the hybridization rate of an acridinium ester probe to thesame level of a probe consisting almost entirely of 2′-O-methylnucleotides (Probe B). In contrast, the T_(m) of a probe containing four2′-O-methyl nucleotides on each side of the acridinium ester linker siteis lower than the T_(m) of a probe:target hybrid in which the probecontains additional 2′-O-methyl nucleotides. Thus, according to thepresent invention it is possible to optimize, or “tune”, the performanceof an acridinium ester labeled probe with respect to its hybridizationrate, differential hydrolysis, and melting properties. For example, anacridinium ester-labeled probe containing four 2′-O-methyl nucleotideson either side of the acridinium ester linker site will have itshybridization rate and differential hydrolysis properties maximallyoptimized, while a hybrid containing this probe will exhibit only asmall increase in its melting temperature.

As substantially contiguous 2′-O-methyl nucleotides are added to replacedeoxyribonucleotides in the labeled probe, the hybridization rate anddifferential hydrolysis properties of the probe:target hybrid willremain substantially constant while its T_(m) will continue to increase.The ability to increase, incrementally, the influence of a probe on theT_(m) of a probe:target hybrid allows one to adjust the specificity of aprobe so as not to cross-react with closely related sequences, as shownin Table 25 above.

Example 20 Effect of Propyne-Modifications on the T_(m) of Probe:TargetHybrids

In order to illustrate the general usefulness of the compositions andmethods of the present invention in the diagnostic application ofnucleic acid hybridization technology, oligonucleotides were constructedhaving a modification other than a 2′-modification to the ribofuranosylmoiety, but which also caused an increase in the binding affinity of aprobe for its target. In this example, oligonucleotides were synthesizedcontaining two nucleotides modified at the nitrogenous base.Specifically,N-diisobutylaminomethylidene-5-(1-propynyl)-2′-deoxycytidine; (acytidine analog) and 5-(1-propynyl)-2′-deoxyuridine) (a thymidineanalog). These nucleotide analogs are commercially available, forexample, from Glen Research in Sterling, Va.

As a first consideration, probes having 22 bases and an acridinium-esterattached at a site located between a thymine base and a guanine base inProbes O and P and between two thymine bases in Probe Q, but containingvarying numbers of propyne-modified nucleotides, were hybridized totarget rRNA in the presence of helper probes to examine the effect ofthe modification on the T_(m) of acridinium ester hybrids. Probe Wcontained no propyne modifications. Probe Q contained two propynemodifications, one directly adjacent to each side of the labelattachment site. Probe P contained 11 propyne modifications, includingfour contiguous modifications directly adjacent and 5′ to the labelattachment site and seven modifications located at bases spaced 3, 4, 6,9-11 and 14 bases away from and 3′ to the label attachment site.

Hybridization and T_(m) determinations were performed as described aboveusing detection of acridinium ester-labeled hybrids. As summarized belowin Table 26, these data indicate that the T_(m) of the oligonucleotide,when hybridized to an RNA target, increased an average of 1° C. forevery replacement of a pyrimidine with a propyne-substituted pyrimidine.

TABLE 26 Probe Propyne Residues T_(m) (chemiluminescent) ΔT/Propyne O  071 — P  2 72 0.5 Q 11 82 1.0

Example 21 Effect of Propyne Modifications on the Hybridization Kineticsof Oligonucleotides

To examine the effect of propyne groups on the hybridization kinetics ofoligonucleotides, the rate of hybridization of the propyne-labeledprobes of Example 20 to RNA were examined by C_(o)t analysis, asdescribed in Example 6. As summarized below in Table 27, the probecontaining two propyne groups (Probe O) hybridized at the same rate asthe probe containing no propyne groups (Probe P), while the probecontaining 11 propyne groups (Probe O) hybridized 1.9-fold faster.

TABLE 27 Probe C_(o)t_(1/2) Relative Rate O 0.75 × 10⁻⁵ 1   P 0.81 ×10⁻⁵  0.93 Q 0.39 × 10⁻⁵ 1.9

These data support the generality of the present invention bydemonstrating that modifications to oligonucleotides which result in anincreased T_(m) also cause the rate of hybridization of the modifiedoligonucleotide to its target to increase compared to an unmodifiedoligonucleotide of the same base sequence. Moreover, this example alsodemonstrates that such modifications may occur in the nitrogenous basemoiety as well as the sugar moiety. Those of skill in the art willrecognize that such modifications may also occur in the internucleotidelinkage as well.

Although the foregoing disclosure describes the preferred embodiments ofthe present invention, Applicant should not be limited thereto. Those ofskill in the art to which this invention. applies will comprehendadditional embodiments in light of this disclosure. Moreover, additionalembodiments are within the claims which conclude this specification andtheir equivalents.

1. A kit comprising: a probe molecule for use in determining the presence of an RNA target in a sample, the probe comprising complementary first and second base regions that form a hybrid containing at least one 2′-O-alkyl ribonucleotide, wherein the probe forms a stable complex with the RNA target but not with a non-targeted nucleic acid under nucleic acid assay conditions, such that the RNA target can be detected, and wherein the complex comprises a single-stranded form of the probe; and a solid support for immobilizing the RNA target so that unbound nucleic acids and other components of the sample can be removed from the RNA target.
 2. The kit of claim 1, wherein the solid support has a magnetic charge.
 3. The kit of claim 1 further comprising: one or more nucleic acid polymerases; nucleotide triphosphates; and one or more amplification oligonucleotides, wherein each of said amplification oligonucleotides is, in the presence of a nucleic acid analyte and under amplification conditions, extended to form part of a nucleic acid extension product containing the RNA target or directs the synthesis of a nucleic acid transcription product containing the RNA target.
 4. The kit of claim 3, wherein the nucleic acid polymerases and amplification oligonucleotides are sufficient to perform a transcription-based amplification reaction.
 5. The kit of claim 1, wherein the first base region contains at least one 2′-O-alkyl ribonucleotide, and wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.
 6. The kit of claim 1, wherein that portion of the first base region which hybridizes to the second base region includes a cluster of at least 4 2′-O-alkyl ribonucleotides.
 7. The kit of claim 6, wherein the probe includes a conjugate molecule joined to the probe at a site located within the cluster of the first base region.
 8. The kit of claim 6, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.
 9. The kit of claim 1, wherein that portion of the first base region which hybridizes to the second base region includes at least one nucleotide which is not a 2′-O-alkyl ribonucleotide.
 10. The kit of claim 9, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.
 11. The kit of claim 1, wherein each nucleotide of that portion of the first base region which hybridizes to the second base region is a 2′-O-alkyl ribonucleotide.
 12. The kit of claim 11, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.
 13. The kit of claim 1, wherein each nucleotide of the probe is a 2′-O-alkyl ribonucleotide.
 14. The kit of claim 1, wherein the first and second base regions form a hybrid that is more stable than a hybrid formed between unmodified forms of the first and second base regions consisting of RNA and/or DNA.
 15. The kit of claim 1, wherein the probe includes a conjugate molecule.
 16. The kit of claim 1, wherein the first and second base regions are contained within an oligonucleotide that is between 10 and 100 bases in length.
 17. The kit of claim 1, wherein the probe comprises a detectable label.
 18. The kit of claim 17, wherein the detectable label comprises a fluorescent molecule.
 19. The kit of claim 1, wherein the RNA target is ribosomal RNA.
 20. The kit of claim 1, wherein the probe forms a stable complex with a region of the RNA target that is folded under the nucleic acid conditions.
 21. The kit of claim 20, wherein the probe includes at least 5 contiguous 2′-O-alkyl ribonucleotides.
 22. The kit of claim 20, wherein the RNA target is ribosomal RNA.
 23. The kit of claim 20, wherein the kit does not include helper probes.
 24. The kit of any one of claims 1 to 23, wherein each 2′-O-alkyl ribonucleotide is a 2′-O-methyl ribonucleotide.
 25. A reaction mixture comprising: one or more amplification oligonucleotides in the presence of at least one nucleic acid polymerase and nucleotide triphosphates sufficient to form an RNA amplification product; and a probe molecule comprising first and second base regions hybridized to each other and having at least one 2′-O-alkyl ribonucleotide, wherein the probe forms a stable and detectable complex with the amplification product but not with non-target nucleic acid, and wherein the complex comprises a single-stranded form of the probe.
 26. The reaction mixture of claim 25, wherein the one or more amplification oligonucleotides and the probe are present in the reaction mixture when the amplification reaction is initiated.
 27. The reaction mixture of claim 25, wherein the first base region contains at least one 2′-O-alkyl ribonucleotide, and wherein the first base region forms a complex with the amplification product.
 28. The reaction mixture of claim 25, wherein that portion of the first base region which hybridizes to the second base region includes a cluster of at least 4 2′-O-alkyl ribonucleotides.
 29. The reaction mixture of claim 28, wherein the probe includes a conjugate molecule joined to the probe at a site located within the cluster of the first base region.
 30. The reaction mixture of claim 28, wherein the first base region forms a complex with the amplification product.
 31. The reaction mixture of claim 25, wherein that portion of the first base region which hybridizes to the second base region includes at least one nucleotide which is not a 2′-O-alkyl ribonucleotide.
 32. The reaction mixture of claim 31, wherein the first base region forms a complex with the amplification product.
 33. The reaction mixture of claim 25, wherein each nucleotide of that portion of the first base region which hybridizes to the second base region is a 2′-O-alkyl ribonucleotide.
 34. The reaction mixture of claim 33, wherein the first base region forms a complex with the amplification product.
 35. The reaction mixture of claim 25, wherein each nucleotide of the probe is a 2′-O-alkyl ribonucleotide.
 36. The reaction mixture of claim 25, wherein the first and second base regions form a hybrid that is more stable than a hybrid formed between unmodified forms of the first and second base regions consisting of RNA and/or DNA.
 37. The reaction mixture of claim 25, wherein the probe includes a conjugate molecule.
 38. The reaction mixture of claim 25, wherein the first and second base regions are contained within an oligonucleotide that is between 10 and 100 bases in length.
 39. The reaction mixture of claim 25, wherein the probe comprises a detectable label.
 40. The reaction mixture of claim 39, wherein the detectable label comprises a fluorescent molecule.
 41. The reaction mixture of claim 25, wherein the the amplification product is formed from a ribosomal RNA.
 42. The reaction mixture of claim 25, wherein the probe forms a stable complex with a folded region of the amplification product.
 43. The reaction mixture of claim 42, wherein the reaction mixture does not include helper probes.
 44. The reaction mixture of claim 42, wherein the probe includes at least 5 contiguous 2′-O-alkyl ribonucleotides.
 45. The reaction mixture of claim 42, wherein the amplification product is formed from a ribosomal RNA.
 46. The reaction mixture of claim 25, wherein the amplification oligonucleotides and the at least one nucleic acid polymerase are sufficient to perform a transcription-based amplification reaction.
 47. The reaction mixture of any one of claims 25 to 46, wherein each 2′-O-alkyl ribonucleotide is a 2′-O-methyl ribonucleotide. 